Novel modified leucine and phenylalanine dipeptides modulate viability and attachment of cancer cells

DOI: 10.1016/j.ejmech.2020.112036

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European Journal of Medicinal Chemistry (2020)

Update date:2022-08-30

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Authors:

Jorda, Radek

Magar, Pratibha

Hendrychová, Denisa

Pauk, Karel

Dibu?, Michal

Pila?ová, Eli?ka

Imramovsky, Ale?

Kry?tof, Vladimír

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Article abstract of DOI:10.1016/j.ejmech.2020.112036

Here, we describe the synthesis and biological characterization of 32 novel phenylalanine and leucine dipeptides modified on both the N and C termini by salicylic acid and aromatic or alicyclic amines, respectively. All compounds displayed antiproliferative activity in the tested cancer cell lines and eight of the compounds exhibited single digit micromolar GI₅₀ values. Treated cells rapidly detached from surface of tissue culture dishes and we found that focal adhesion kinase (FAK), p130CAS and paxillin, which are important regulators of cell adhesion, were dephosphorylated at Y397, Y410 and Y118, respectively. The most potent compound reduced proliferation in the HCT-116 cell line in a dose-dependent manner, as shown by a decrease in 5-bromo-2′-deoxyuridine incorporation into DNA. Furthermore, this compound increased the levels of several apoptotic markers, including activated caspases, and increased site-specific poly-(ADP-ribose)polymerase (PARP) cleavage.

Full text of DOI:10.1016/j.ejmech.2020.112036

European Journal of Medicinal Chemistry 188 (2020) 1 12036

Contents lists available at ScienceDirect

European Journal of Medicinal Chemistry

journal homepage: h ttp://www.elsevier.com/locate/ejmech

Research paper

Novel modiﬁed leucine and phenylalanine dipeptides modulate

viability and attachment of cancer cells

, e

ꢀ ^a

Radek Jorda ^{a 1}, Pratibha Magar ^c, Denisa Hendrychova , Karel Pauk , Michal Dibus

ꢁ ^d

a, b, *

ꢁ

ꢀ ^c

ꢁ

Eliska Pilarova , Ales Imramovský

, Vladimír Krystof

ꢁ

ꢂ

Laboratory of Growth Regulators, Palacky University & Institute of Experimental Botany, The Czech Academy of Sciences, Slechtitelu 27, 78371, Olomouc,

Czech Republic

ꢁ

ꢀ

Institute of Molecular and Translational Medicine, Faculty of Medicine and Dentistry, Palacký University, Hnevotínska 5, 77900, Olomouc, Czech Republic

ꢀ

Institute of Organic Chemistry and Technology, Faculty of Chemical Technology, University of Pardubice, Studentska 95, 532 10, Pardubice, Czech Republic

ꢁ ꢀ

Department of Cell Biology, Charles University, Vinicna 7, 12800, Prague, Czech Republic

ꢂ

ꢀ

Biotechnology and Biomedicine Centre of the Academy of Sciences and Charles University (BIOCEV), Prumyslova 595, 25242, Vestec u Prahy, Czech

Republic

a r t i c l e i n f o

a b s t r a c t

Article history:

Here, we describe the synthesis and biological characterization of 32 novel phenylalanine and leucine

dipeptides modiﬁed on both the N and C termini by salicylic acid and aromatic or alicyclic amines,

respectively. All compounds displayed antiproliferative activity in the tested cancer cell lines and eight of

the compounds exhibited single digit micromolar GI₅₀values. Treated cells rapidly detached from surface

of tissue culture dishes and we found that focal adhesion kinase (FAK), p130CAS and paxillin, which are

important regulators of cell adhesion, were dephosphorylated at Y397, Y410 and Y118, respectively. The

most potent compound reduced proliferation in the HCT-116 cell line in a dose-dependent manner, as

shown by a decrease in 5-bromo-2⁰-deoxyuridine incorporation into DNA. Furthermore, this compound

increased the levels of several apoptotic markers, including activated caspases, and increased site-

speciﬁc poly-(ADP-ribose)polymerase (PARP) cleavage.

Received 1 October 2019

Received in revised form

21 December 2019

Accepted 3 January 2020

Available online 7 January 2020

Keywords:

cytotoxicity

Dipeptide

Apoptosis

Adhesion

Detachment

1. Introduction

properties being somewhere in between.

Existing anticancer peptides have been classiﬁed according to

Cytotoxic chemotherapy is a major approach to treat cancer and

saves many lives each year, but it has several drawbacks, especially

due to the side effects related to low tumour selectivity and the

development of drug resistance. Recently, targeting active onco-

proteins or cancer-speciﬁc features in general using rationally

designed drugs has translated into clinical practice and become a

standard option in many indications. Such drugs are commonly

based on small molecules (mostly interacting with cellular targets)

or biological macromolecules (usually restricted to extracellularly

exposed targets), along with peptides, due to their size and

their utilization; for example, carriers of radionuclides or cytotoxic

drugs, hormone agonists and antagonists, modulators of the im-

mune response, angiogenesis and adhesion, or peptides targeting

cellular processes [1e3]. Similar to other groups, this last group

comprises peptides with a variety of mechanisms, including inhi-

bition of signal transduction pathways, triggering mitochondrial

depolarization, altering gene expression or blocking protein

degradation [3,4].

A major advantage of peptide drug discovery is the wide avail-

ability of variable building blocks, which are deﬁnitely not limited

to proteinogenic amino acids. In fact, the presence of non-canonical

amino acids or chemical modiﬁcation of their functional groups

could increase the afﬁnity and selectivity of peptides and overcome

some of their signiﬁcant drawbacks, such as their stability in vivo

due to proteolytic degradation [5,6].

* Corresponding author. Laboratory of Growth Regulators, Palacky University &

ꢁ

ꢂ

Institute of Experimental Botany, The Czech Academy of Sciences, Slechtitelu 27,

78371, Olomouc, Czech Republic.

** Corresponding author.

In our previous studies, modiﬁed dipeptides and tripeptides

were reported to disrupt the dynamics of the actin cytoskeleton or

inhibit the proteasome [7e10]. The N-terminus of these peptides

E-mail addresses: ales.imramovsky@upce.cz (A. Imramovský), vladimir.krystof@

ꢁ

upol.cz (V. Krystof).

These authors contributed equally.

https://doi.org/10.1016/j.ejmech.2020.1 12036

R. Jorda et al. / European Journal of Medicinal Chemistry 188 (2020) 112036

were bound to O-benzyl salicylic acid, which has already been

incorporated into various compounds with antimicrobial, antipar-

asitic and anticancer activities [11e15]. In this study, we investi-

gated novel phenylalanine and leucine dipeptides modiﬁed on the

N terminus by salicylic acid. The C terminus of these compounds

was modiﬁed by substitution with aromatic amines containing

electron donating or electron withdrawing substituents or by

alicyclic or heterocyclic amines (Scheme 1). The compounds

showed antiproliferative activity in cancer cell lines in vitro with

micromolar GI₅₀values, but this was not due to inhibition of the

proteasome as described for the related compounds [9]. We

observed that the treated cells exhibited an unusual detachment

from the surface, and we hypothesized that these compounds can

modulate adhesion-related processes. Our results indeed revealed

that the compounds interfered with FAK signalling, which is

important for cell adhesion.

triﬂuoromethyl phenyl were also synthesized and investigated to

conﬁrm the inﬂuence of the dipeptide scaffold on the ﬁnal

molecules.

The synthesis of the investigated compounds is described in

Scheme 1; synthesis began with the preparation of 5-chloro-O-

benzyl-salicylic acid (1) according to the literature [16]. This com-

pound was coupled with a free base of a L-leucine or L-phenylal-

anine methyl ester under peptide coupling conditions (N-(3-

dimethylaminopropyl)-N⁰-ethylcarbodiimide

hydrochloride,

EDCI∙HCl; 1-hydroxybenzotriazole hydrate; HOBt hydrate) in

dichloromethane (DCM) at ambient temperature. Isolated amide 3

(69e87% yield) was subjected to basic hydrolysis of the ester in an

aqueous medium (water:1,4-dioxane, 1:1) to directly produce an

excellent yield of acid 4 (88e98%). The isolation of this product

from the reaction mixture was simple and did not require chro-

matographic puriﬁcation. These acids were the starting material for

the synthesis of dipeptide ester 5 via the formation of another

amidic bond. The conditions, solvents, isolation and yield were

similar to those in the formation of compound 3. The key inter-

mediate acids 6 were obtained via basic hydrolysis at high yields

(68e84%). The introduction of a second stereogenic center brought

the possibility of easy detection of possible racemization using 1H

NMR measurements in compounds 5 or 6. The removal of unfa-

vorable diastereoisomer was speciﬁc for each compound. For

example, for the L-Phe-L-Leu esters 5X, the optimal separation of

diastereoisomers was performed by crystallization from an ethyl

acetateehexane mixture. The ﬁltrate contained the optically pure

compound, while the crystals were enriched with the diastereo-

isomer mixture (based on ¹H NMR measurements). Different ap-

2. Results and discussion

2.1. Chemistry

The concept of the synthesis in this study was based on the

modiﬁcation of the L-Leu-L-Leu-OH amide of O-benzyl-protected 5-

chloro-salicylic acid [7]. This compound was modiﬁed to an amide

with various aromatic, alicyclic and heterocyclic amines. The

phenolic hydroxyl group was then liberated by hydrogenolysis to

conﬁrm whether the free phenolic hydroxyl group inﬂuenced

biological properties. Biological screening showed interesting

properties for the compounds with electron withdrawing sub-

stituents (3,4-dichloro phenyl and triﬂuoromethyl phenyl). There-

proaches were used for the puriﬁcation of L-Leu-L-Leu dipeptide

acid 6a-d. The best solution resulted from the crystallization of the

ﬁnal acid from the ethyl acetateehexane mixture. The optically

pure acid 6a-d was obtained as a white crystal compound. A

fore, the library of prepared compounds was extended to include

phenylalanine and its combination with L-leucine. Sample com-

pounds containing only one amino acid ( -Phe or -Leu) with

L-

Scheme 1. Reagent and conditions for synthesis of novel salicylamides: a ¼ EDCI∙HCl (0.97 eq), HOBt (1.02 eq), DIPEA (1.0 eq), DCM, RT, 2e18 h b ¼ LiOH, dioxan-water 1:1, 50 ^ꢀC,

1 h, c ¼ H₂/Pd, EtOAc, RT, 24 h.

R. Jorda et al. / European Journal of Medicinal Chemistry 188 (2020) 112036

subsequent carbodiimide-mediated reaction was performed with

selected anilines in dry dichloromethane as solvents to produce

compounds 7a-n. The phenolic hydroxyl group was then liberated

using hydrogenolysis (H₂/Pd). The obtained triamides 8a-n were

isolated at low to excellent isolated yields (35e85%). All prepared 7

and 8 compounds were characterized as original organic com-

pounds by melting points, ¹H and ¹³C NMR, IR, elemental analysis

and Maldi-TOF analysis (for spectra and additional information,

please see Supplementary information).

We then analysed the effect of the eight most potent com-

pounds on the proliferation and cytotoxicity of HCT-116 cells by

live-cell imaging. As shown in Fig.1A, most compounds induced the

cytotoxicity of HCT-116 cells approximately 12 h after the com-

pound was added. Compounds 8e and 8j were the only derivatives

that did not increase cell death compared to the untreated control

(Fig. 1A). The most potent compound 8l signiﬁcantly induced cell

death early in the treatment period. In the next experiment, we

showed that 8l decreased the proliferation of HCT-116 cells in a

dose-dependent manner over 24 h of incubation (see Fig. 1B).

To verify the importance of the dipeptide moiety, four novel

salicylamides were synthesized. These compounds contained only

L-Phe or L-Leu and phenyl amide moieties with 4- triﬂuoromethyl

substituents. The phenolic hydroxyl group was then liberated using

hydrogenolysis (H2/Pd). The synthesis of these compounds is

shown in Scheme 2.

2.3. Compound 8l causes cell detachment

We observed that HCT-116 cells exhibited visible changes in

their morphology after 24 h of treatment with high doses of 8l; the

cells became round and mostly detached from the surface

(Supplementary Fig. S1). We hypothesized that 8l can modulate the

attachment of cells. The disruption of contacts between integrins

and the extracellular matrix usually results in reduced FAK sig-

nalling, which leads to the detachment of cells. Therefore, we

analysed phosphorylations of FAK, p130Cas and paxillin, which are

key components of integrin control and integrin-mediated cyto-

skeletal reorganization. As shown in Fig. 2A, treatment with 8l

caused the dephosphorylation of all three proteins in HCT-116 cells

in a dose-dependent manner. Importantly, the dephosphorylation

of FAK at Y397 was observed at a much earlier time points (Fig. 2B)

than when the detached cells were observed.

2.2. Antiproliferative activity of novel dipeptides

All compounds were tested for their antiproliferative properties

against acute lymphoblastic leukaemia CEM and chronic myelog-

enous leukaemia K562 cell lines; the data are presented in Table 1.

The results clearly show that most compounds display GI₅₀values

in the mid-micromolar range. An interesting trend was observed

for compounds with free phenolic groups as a result of deprotection

by hydrogenolysis; the antiproliferative properties of these com-

pounds were signiﬁcantly improved compared with those of O-

benzyl protected compounds, and some derivatives showed up to

single-digit micromolar values. Only derivatives containing cyclo-

hexyl (7f and 8f) and morpholine (7h and 8h) moieties did not

follow this trend. Among the deprotected salicylamides (with two

incorporated leucines in the peptide part) bearing different elec-

tron withdrawing or electron accepting groups in the amide part of

the molecules, only derivatives bearing 3,4-dichlorophenyl and 4-

triﬂuorophenyl moieties proved to potently inhibit the investi-

gated cell lines at single-digit micromolar concentrations

2.4. Effect of 8l on proliferation and cell death

Flow cytometry analysis of HCT-116 cells treated with different

concentrations of 8l showed a gradual decrease in actively prolif-

erating cells (BrdU-positive) without the dominant accumulation in

any phase of the cell cycle; however, the subdiploid population

showed ongoing cell death (Fig. 3A).

(GI₅₀¼ 2.6e7.9

mM). Only derivatives bearing these terminal sub-

stituents were further investigated for the optimal combination of

amino acid side chains (leucine or phenylalanine) in the peptide

part of the molecules. A distinct effect was observed for only a se-

ries of O-benzyl protected salicylamides, and we showed that

compounds bearing two aromatic phenylalanine side chains were

The effect of 8l on the expression of apoptosis-related proteins

in HCT-116 was evaluated by immunoblotting and by biochemical

determination of caspase activity. As shown in Fig. 3B, we detected

the fragments of activated caspase-7 as well as the dose-dependent

cleavage of poly(ADP-ribose) polymerase (PARP), which is a typical

marker of apoptosis. Ongoing apoptosis was also conﬁrmed by the

increased activities of caspase-3 and caspase-7, as quantiﬁed with a

biochemical assay (Fig. 3C). Furthermore, we monitored the

expression of anti-apoptotic proteins involved in the mitochondrial

pathway of apoptosis (XIAP, Bcl-xl and Bcl-2), but no signiﬁcant

changes were observed (Fig. 3B). Interestingly, we observed slight

fragmentation of the autophagy-related protein LC3 (microtubule-

associated protein 1-light chain 3), which suggested the ability of 8l

to simultaneously induce autophagy and apoptosis in treated cells.

signiﬁcantly more potent than other derivatives containing both

L-

Phe and -Leu side chains (compare 7k with 7e, 7i, 7j and 7n with

7m, 7l, 7d). Conversely, the antiproliferative properties of the

deprotected counterparts were not dependent on the type of

incorporated amino acids.

The antiproliferative activity of the most potent derivatives was

further tested against a panel of human cancer cell lines derived

from solid cancers, including melanoma (G361), breast cancer

(MCF-7) and colorectal cancer (HCT-116); in all cases, cytotoxicity

retained single-digit micromolar values (Table 2).

Scheme 2. Preparation of modiﬁed dipeptides with one aminoacid (9a, 9b) and their deprotected variants (10a and 10b). a ¼ EDCI∙HCl (0.97 eq), HOBt (1.02 eq), DIPEA (1.0 eq),

DCM, RT, 2e18 h, c ¼ H₂/Pd, EtOAc, RT, 24 h.

R. Jorda et al. / European Journal of Medicinal Chemistry 188 (2020) 112036

Table 1

Antiproliferative activities of novel salicylamides.

GI₅₀(m

M)^a

Compound

R¹

R²

R³

R⁴

K562

CEM

i-Pr

4-CH₃O-Ph

4-CH₃-Ph

68.3

87.7

71.2

74.6

>100

19.8

79.2

21.0

57.5

63.5

9.9

73.3

61.7

12.7

51.0

30.7

20.1

7.5

53.2

52.8

48.9

59.9

56.4

9.8

34.3

20.1

45.4

55.9

32.8

48.1

56.7

11.3

23.3

13.2

10.2

6.2

4-CF₃-Ph

3,4-Di-Cl-Ph

cyclohexyl

thiazol-2-yl

morpholin-1-yl

3,4-Di-Cl-Ph

4-CF₃-Ph

4-CH₃O-Ph

4-CH₃-Ph

i-Pr

10a

10b

i-Pr

4-CF₃-Ph

3,4-Di-Cl-Ph

cyclohexyl

thiazol-2-yl

morpholin-1-yl

3,4-Di-Cl-Ph

4-CF₃-Ph

3.2

2.6

55.7

31.1

>50

5.1

3.7

5.8

6.3

5.1

38.2

19.7

54.9

3.3

2.6

3.1

7.9

3.5

4.7

>50

5.0

35.4

31.5

5.0

i-Pr

4-CF₃-Ph

5.2

4.5

Measured at least in triplicates. Data including SD values are available from SI.

Table 2

Antiproliferative activities of the most potent salicylamides.

modulate the attachment of cancer cells and FAK signalling, which

is important for cell survival, proliferation and migration. FAK is

emerging as a promising anticancer drug target because it is

hyperactivated in many solid tumours, and its increased expression

and activity are associated with poor clinical outcomes [17,18]; the

anticancer effects of salicylamides are therefore worth further

investigation.

Compound

GI₅₀

G361

M)^a

MCF-7

HCT-116

11.5 3.1

5.2 0.9

5.4 0.7

4.8 1.1

4.5 0.5

6.3 0.9

8.1 2.1

5.1 0.14

5.3 0.1

4.1 1.5

7.3 0.01

4.6 0.4

5.2 0.2

5.5 0.01

3.6 0.2

5.6 0.9

5.8 0.2

4.0 0.3

4.4 2.1

2.8 0.2

3.0 0.5

5.3 0.5

3.2 0.2

4.5 2.0

3. Experimental part

3.1. Chemistry

Measured at least in triplicates; n.t. - not tested.

All reagents and solvents were purchased from commercial

sources (Sigma-Aldrich, Merck, Acros Organics, J & K Scientiﬁc).

Commercial grade reagents were used without further puriﬁcation.

Reactions were monitored by thin layer chromatography plates

coated with 0.2 mm silica gel 60 F254 (Merck). TLC plates were

visualized with UV (254 nm) in a 5% solution of phosphomolybdic

acid in ethanol. All melting points were determined on a Melting

Point B-540 apparatus (Büchi, Switzerland) and are uncorrected.

The IR spectra were recorded on a Nicolet 6700 FT-IR spectrometer

(Thermo Fisher Scientiﬁc, Wal-tham, MA, USA) over the range of

400e4000 cm^ꢁ1using the ATR technique. The NMR spectra were

measured in dimethyl sulfoxide (DMSO) solutions at room tem-

perature on a Bruker Avance™ III 400 spectrometer at the fre-

quencies 400 MHz (¹H) and 100.26 MHz (¹³C) or on a Bruker

2.5. Conclusion

Our previous studies revealed that salicylamide-based com-

pounds potently induce apoptosis in different cancer cell lines [7,8].

In this article, we described new derivatives of O-benzyl-5-chloro-

salicylicamides with integrated L-phenylalanines or with L-leucines

where the amide part was modiﬁed by different electron donating

or electron withdrawing substituents. The antiproliferative activity

was signiﬁcantly improved by the deprotection of compounds by

hydrogenolysis, which resulted in derivatives that potently inhibi-

ted the proliferation of different cancer cell lines at micromolar

doses. Further experiments revealed that these salicylamides can

R. Jorda et al. / European Journal of Medicinal Chemistry 188 (2020) 112036

Fig. 1. The time-course effect of most active compounds in HCT-116 cells. (A) For evaluation of relative cytotoxicity the cells were treated with 25

M compounds for 24 h, and data

were acquired with a live-cell imaging microscope. IncuCyte® Cytotox Red Reagent was used for the real-time evaluation of cell membrane integrity/cell death. (B) The cell

proliferation of HCT-116 cells treated with different concentrations of 8l was quantiﬁed by calculating the occupied area (% of conﬂuence) over time up to 24 h.

Ascend™ 500 spectrometer at frequencies of 500.13 MHz (¹H) and

125.76 MHz (¹³C{1H}). The chemical shifts,

d, are given in ppm,

relative to the residual solvent peaks (CDCl₃- 7.26 ppm, DMSO e

2.5 ppm). Elemental analyses (C, H, N) were performed on an

automatic Flash 2000 Organic elemental microanalyser for all

compounds with exception of 8f, for which the purity was deter-

mined by HPLC-photo diode array detector and gave satisfactory

results (89%). Mass spectrometry with high resolution was deter-

mined by the “dried droplet” method using a MALDI mass spec-

trometer LTQ Orbitrap XL (Thermo Fisher Scientiﬁc) equipped with

a nitrogen UV laser (337 nm, 60 Hz).

3.2. Synthesis and characterization of intermediates

All intermediates (1, 2a-b, 3a-b, 4, 5a-d and 6a-d) were syn-

thesized and characterized according to our previous approaches

[7,8].

3.3. General procedure and characterization of benzyl-protected

amides 7a-n

EDCI∙HCl (0.97 eq) and HOBt hydrate (1.02 eq) were added to a

solution of acid 6 (1.0 eq) in dry DCM (1 mmol per 50 ml) at room

temperature. After 30 min, a solution of the appropriate aniline

(1.0 mmol) in dry DCM (1 mmol per 15 ml) was added for 2 min. The

mixture was stirred at room temperature under an N₂atmosphere

until all starting materials were consumed (usually 2e16 h). Af-

terwards, the reaction mixture was concentrated under reduced

pressure, dissolved in ethyl acetate (30 ml per 1 mmol) and

extracted with water (2 ꢂ 10 ml per 1 mmol), sat. solution of

NaHCO₃(2 ꢂ 10 ml per 1 mmol), 5% citric acid solution (2 ꢂ 10 ml

per 1 mmol), brine (1 ꢂ 10 ml per 1 mmol) and water (1 ꢂ 10 ml per

1 mmol). The organic extract was dried with Na₂SO₄and ﬁltered,

and the solvent was distilled off under vacuum. The residue was

puriﬁed by column chromatography (silica gel eluted with 25%

ethyl acetate in n-hexane) to give the benzylated product 7. In most

cases, the obtained compound was crystallized from an n-hexane/

ethyl acetate mixture. Products 7a-n were obtained at a 49e80%

isolated yield.

Fig. 2. 8l induces dephosphorylation of proteins involved in FAK signalling in HCT-

116 cells in a dose (A) and time-dependent manner (B). Adhered cells (48 h post

plating) were treated as indicated and proteins were then analysed by immunoblot-

ting.

b-actin and GAPDH were probed for equal protein loading. Asterisk indicates

sample corresponding to visibly detached cells at the end of experiment.

R. Jorda et al. / European Journal of Medicinal Chemistry 188 (2020) 112036

Fig. 3. 8l induces cell death in HCT-116 cells. (A) Effect of 8l on the cell cycle was analysed by ﬂow cytometry (10 000 counts, DNA stained by propidium iodide and 5-bromo-2⁰-

deoxyuridine). The number in the bottom right corner refers to the percentage of apoptotic cells (subG1 population). (B) Immunoblotting analysis of cells treated with 8l for 24 h.

tubulin was included as a control for protein loading. (C) Relative caspase-3/7 activity in lysates of cells treated with 8l. The activities of the caspases were measured using the

ﬂuorogenic substrate Ac-DEVD-AMC and normalized to an untreated control.

3.3.1. Characterization of 2-benzyloxy-5-chloro-N-((2S)-1-(((2S)-1-

(4-methoxy)phenyl)amino)-4-methyl-1-oxopentan-2-yl)amino)-4-

methyl-1-oxopentan-2-yl)benzamide 7a

3.3.2. Characterization of 2-benzyloxy-5-chloro-N-((2S)-1-(((2S)-

1-(4-methyl)phenyl)amino)-4-methyl-1-oxopentan-2-yl)amino)-4-

methyl-1-oxopentan-2-yl)benzamide 7b

White solid; yield 70%; mp 182e184 ^ꢀC; R_{f (hex/EtOAc e 3/1)}¼ 0.12.

White crystalline solid; yield 75%; mp 178e182 ^ꢀC; R_{f (hex/EtOAc e}

IR (ATR): 3269, 3068, 2929, 1697, 1683, 1599, 1463, 1244, 1123, 750,

¼ 0.21. IR (ATR): 3280, 3067, 2927, 1606, 1596, 1513, 1367, 1270,

3/1)

695 cm^ꢁ1. ¹H NMR (400 MHz, DMSO‑d₆):

9.79 (1H, s, NH-Ph), 8.33

1120, 750, 695 cm^ꢁ1. ¹H NMR (400 MHz, DMSO‑d₆):

d 9.83 (1H, s,

(1H, d, J ¼ 7.6 Hz, NH-CH-CH₂-CH-(CH₃)₂), 8.18 (1H, d, J ¼ 8.4 Hz,

NH-CH-CHH-CH-(CH₃)₂), 7.74 (1H, d, J ¼ 2.8 Hz, Ar-H), 7.58e7.51

(5H, m, Ar-H), 7.41e7.32 (4H, m, Ar-H), 6.88 (2H, d, J ¼ 9.2 Hz, Ar-H),

5.26 (2H, s, O-CH₂-Ph), 4.50e4.42 (2H, m, NH-CH-CH₂-CH-(CH₃)₂),

NH-CH-CH₂-CH-(CH₃)₂), 3.71 (3H, s, O-CH₃), 1.63e1.51 (3H, m, NH-

CH-CH₂-CH-(CH₃)₂), NH-CH-CHH-CH-(CH₃)₂), 1.40 (2H, t, J ¼ 6.4 Hz

NH-CH-CHH-CH-(CH₃)₂), 1.26 (1H, t, J ¼ 8.4 Hz, NH-CH-CH₂-CH-

(CH₃)₂), 0.89 (6H, dd, J ¼ 6.4 Hz, J ¼ 19.6 Hz, NH-CH-CH₂-CH-

(CH₃)₂), 0.73 (6H, dd, J ¼ 5.6 Hz, J ¼ 27.6 Hz, NH-CH-CH₂-CH-(CH₃)₂).

NH-Ph), 8.33 (1H, d, J ¼ 7.6 Hz, NH-CH-CH₂-CH-(CH₃)₂), 8.20 (1H, d,

J ¼ 8 Hz, NH-CH-CH₂-CH-(CH₃)₂), 7.75 (1H, d, J ¼ 2.8 Hz, Ar-H),

7.58e7.49 (5H, m, Ar-H), 7.38e7.32 (4H, m, Ar-H), 7.10 (2H, d,

J ¼ 7.2 Hz, Ar-H), 5.26 (2H, s, O-CH₂-Ph), 4.49e4.45 (2H, m, NH-CH-

CH₂-CH-(CH₃)₂), NH-CH-CH₂-CH-(CH₃)₂), 2.24 (3H, s, CH₃-Ph),

1.64e1.51 (3H, m, NH-CH-CH₂-CH-(CH₃)_2,NH-CH-CHH-CH-(CH₃)₂),

1.41e1.36 (2H, t, J ¼ 6.4 Hz, NH-CH-CHH-CH-(CH₃)₂, NH-CH-CHH-

CH-(CH₃)₂), 1.26 (1H, dd, J ¼ 8 Hz, J ¼ 9.6 Hz, NH-CH-CH₂-CH-

(CH₃)₂), 0.92e0.83 (6H, m, CH₃-CH-CH₃), 0.76 (3H, d, J ¼ 6 Hz, CH₃-

¹³C NMR (100.62 MHz):

172.2, 171.0, 164.0, 155.9, 155.7, 136.4,

CH-CH₃), 0.69 (3H, d,

5.6 Hz, CH₃-CH-CH₃). ¹³C NMR

132.7, 132.6, 130.5, 129.2, 129.0, 128.9, 125.4, 125.0, 121.4, 116.3,

114.5, 71.5, 55.8, 52.5, 52.3, 41.5, 41.4, 25.0, 24.80, 23.64, 23.60, 22.2,

22.0. CHN analysis: Calc. for C₃₃H₄₀ClN₃O₅(594.14): C, 66.71; H,

6.79; N, 7.07. Found: C, 66.80 0.02; H, 6.93 0.02; N, 6.98 0.02.

HRMS: m/z calc. for C₃₃H₄₀ClN₃O₅: 594.27293 [MþH]^þ; 616.25487

[MþNa]^þ; found: 594.27419 [MþH]^þ; 616.25606 [MþNa]^þ.

(100.61 MHz): 172.3, 171.3, 164.0, 155.7, 137.0, 136.4, 132.9, 132.6,

130.6, 129.7, 129.2, 129.0, 128.9, 125.4, 125.0, 119.9, 116.3, 71.5, 52.5,

52.4, 41.5, 41.4, 25.0, 24.8, 23.6, 22.2, 22.0, 21.1, 21.07. CHN analysis:

Calc. for C₃₃H₄₀ClN₃O₄(578.14): C, 68.56; H, 6.97; N, 7.27. Found: C,

68.71 0.02; H, 7.04 0.01; N, 7.19 0.02. HRMS: m/z calc. for

₃₃H₄₀ClN₃O₄: 578.27801 [MþH]^þ; 600.25996 [MþNa]^þ; found:

578.27941 [MþH]^þ; 600.26128 [MþNa]^þ.

R. Jorda et al. / European Journal of Medicinal Chemistry 188 (2020) 112036

3.3.3. Characterization of 2-benzyloxy-5-chloro-N-((2S)-1-(((2S)-

1-(phenyl)amino)-4-methyl-1-oxopentan-2-yl)amino)-4-methyl-1-

oxopentan-2-yl)benzamide 7c

170.4, 165.5, 155.8, 138.0, 134.6, 133.6, 132.6, 132.3, 130.4, 129.7,

129.4, 128.8, 127.5, 127.2, 121.7, 121.6, 119.3, 114.2, 72.3, 53.7, 52.7,

39.8, 39.8, 25.2, 25.0, 23.2, 23.0, 21.6. CHN analysis: Calc. for

White solid; yield 66%; mp 164e168 ^ꢀC; R_{f (hex/EtOAc e 3/1)}¼ 0.18.

C₃₂H₃₆Cl₃N₃O₄(633.00): C, 60.72; H, 5.73; N, 6.64. Found: C,

IR (ATR): 3280, 3066, 2955, 1639, 1596, 1498, 1309, 1270, 1121, 752,

61.04 0.02; H, 5.78 0.02; N, 6.34 0.02. HRMS: m/z calc. for

692 cm^ꢁ1. ¹H NMR (400 MHz, DMSO‑d₆):

9.94 (1H, s, NH-Ph), 8.33

₃₂H₃₆Cl₃N₃O₄: 632.18442 [MþH]^þ; 654.16636 [MþNa]^þ; found:

(1H, d, J ¼ 7.6 Hz, NH-CH-CH₂-CH-(CH₃)₂), 8.22 (1H, d, J ¼ 8.4 Hz,

NH-CH-CH₂-CH-(CH₃)₂), 7.75 (1H, d, J ¼ 2.8 Hz, Ar-H), 7.62 (2H, d,

J ¼ 7.6 Hz, Ar-H), 7.58e7.52 (3H, m, Ar-H), 7.40e7.28 (6H, m, Ar-H),

7.05 (1H, t, J ¼ 7.6 Hz, Ar-H), 5.26 (2H, s, O-CH₂-Ph), 4.51e4.46 (2H,

m, NH-CH-CH₂-CH-(CH₃)₂, NH-CH-CH₂-CH-(CH₃)₂), 1.65e1.52 (3H,

m, NH-CH-CH₂-CH-(CH₃)₂, NH-CH-CHH-CH-(CH₃)₂), 1.42e1.39 (2H,

m, NH-CH-CHH-CH-(CH₃)₂), 1.29e1.24 (1H, m, NH-CH-CHH-CH-

(CH₃)₂), 0.93e0.84 (6H, m, CH₃-CH-CH₃), 0.73 (6H, dd, J ¼ 6 Hz,

632.18520 [MþH]^þ; 654.16725 [MþNa]^þ.

3.3.6. Characterization of 2-benzyloxy-5-chloro-N-((2S)-1-(((2S)-

1-(cyclohexyl)amino)-4-methyl-1-oxopentan-2-yl)amino)-4-

methyl-1-oxopentan-2-yl)benzamide 7f

White solid; yield 74%; mp 172e175 ^ꢀC. IR (ATR): 3279, 3067,

2930, 1596, 1537, 1498, 1300, 1244, 1122, 729, 695 cm^ꢁ1¹H NMR

(400 MHz, DMSO‑d₆):

8.32 (1H, d, J ¼ 7.6 Hz, NH-cyclohexyl), 7.99

J ¼ 28 Hz CH₃-CH-CH₃). ¹³C NMR (100.61 MHz):

172.3, 171.6, 164.0,

(1H, d, J ¼ 8.4 Hz, NH-CH-CH₂-CH-(CH₃)₂), 7.75 (1H, d, J ¼ 2.8 Hz,

NH-CH-CH₂-CH-(CH₃)₂), 7.58e7.53 (4H, m, Ar-H), 7.42e7.33 (4H, m,

Ar-H), 5.26 (2H, s, O-CH₂-Ph), 4.40e4.35 (1H, m, NH-CH-CH₂-CH-

(CH₃)₂), 4.29 (1H, dd, J ¼ 8.4 Hz, J ¼ 15.2 Hz, NH-CH-CH₂-CH-

(CH₃)₂), 3.35 (1H, s, NH-CH cyclohexyl), 1.67 (4H, ABs, NH-CH-CH₂-

CH-(CH₃)₂, NH-CH-CH₂-CH-(CH₃)₂), 1.56e1.53 (2H, m, NH-CH-CH₂-

CH-(CH₃)₂, NH-CH-CH₂-CH-(CH₃)₂), 1.46e1.36 (4H, m, cyclohexyl),

1.29e1.11 (6H, m, cyclohexyl), 0.88e0.80 (6H, m, CH₃-CH-CH₃),

155.7, 139.5, 136.4, 132.6, 130.6, 129.4, 129.2, 129.0, 128.9, 125.4,

125.0,124.0, 119.9, 116.3, 71.5, 52.4, 52.4, 41.5, 41.3, 25.0, 24.8, 23.64,

23.60, 22.2, 22.1. CHN analysis: Calc. for C₃₂H₃₈ClN₃O₄(564.11): C,

68.13; H, 6.79; N, 7.45. Found: C, 68.44 0.02; H, 6.91 0.01; N,

7.38 0.02. HRMS: m/z calc. for C₃₂H₃₈ClN₃O₄: 564.26236 [MþH]^þ;

586.24431 [MþNa]^þ; found: 564.26345 [MþH]^þ; 586.24537

[MþNa]^þ.

0.78e0.68 (6H, m, CH₃-CH-CH₃). ¹³C NMR (100.61 MHz):

d 171.9,

3.3.4. Characterization of 2-benzyloxy-5-chloro-N-((2S)-1-(((2S)-

1-(4-triﬂuoromethyl)phenyl)amino)-4-methyl-1-oxopentan-2-yl)

amino)-4-methyl-1-oxopentan-2-yl)benzamide 7d

171.3, 164.0, 155.7, 136.4, 132.6, 130.5, 129.2, 129.0, 129.0, 125.4,

124.9, 116.3, 71.5, 52.7, 51.5, 48.0, 41.4, 41.4, 32.9, 25.9, 25.1, 24.9,

24.8, 23.6, 23.5, 22.3, 22.1. CHN analysis: Calc. for C₃₂H₄₄ClN₃O₄

White crystalline solid; yield 68%; mp 189e192 ^ꢀC; R_{f (hex/EtOAc e}

(570.16): C, 67.41; H, 7.78; N, 7.37. Found: C, 67.62

0.03; H,

¼ 0.17. IR (ATR): 3268, 3068, 2957, 1682, 1633, 1599, 1377, 1256,

7.91

0.02; N, 7.29

0.02. HRMS: m/z calc. for C₃₂H₄₄ClN₃O₄:

3/1)

1109, 720, 695 cm^ꢁ1. ¹H NMR (400 MHz, DMSO‑d₆):

10.39 (1H, s,

570.30931 [MþH]^þ; 592.29126 [MþNa]^þ; found: 570.30995

NH-Ph), 8.35 (2H, dd, J ¼ 8 Hz, J ¼ 13.6 Hz, NH-CH-CH₂-CH-(CH₃)₂,

NH-CH-CH₂-CH-(CH₃)₂), 7.88 (2H, d, J ¼ 8.4 Hz, Ar-H), 7.79 (1H, d,

J ¼ 2.8 Hz, Ar-H), 7.73 (2H, d, J ¼ 8.8 Hz, Ar-H), 7.62e7.56 (3H, m, Ar-

H), 7.44e7.37 (4H, m, Ar-H), 5.31 (2H, s, O-CH₂-Ph), 4.58e4.51 (2H,

m, NH-CH-CH₂-CH-(CH₃)₂, NH-CH-CH₂-CH-(CH₃)₂), 1.73e1.53 (3H,

m, NH-CH-CH₂-CH-(CH₃)₂, NH-CH-CHH-CH-(CH₃)₂), 1.50e1.43 (2H,

m, NH-CH-CHH-CH-(CH₃)₂), 1.36e1.27 (1H, m, NH-CH-CH₂-CH-

(CH₃)₂), 0.97 (3H, d, J ¼ 6.4 Hz, CH₃-CH-CH₃), 0.92 (3H, d, J ¼ 6.4 Hz,

CH₃-CH-CH₃), 0.82 (3H, d, J ¼ 6 Hz, CH₃-CH-CH₃), 0.75 (3H, d,

[MþH]^þ; 592.29183 [MþNa]^þ.

3.3.7. Characterization of 2-benzyloxy-5-chloro-N-((2S)-1-(((2S)-

1-(thiazol-2-yl)amino)-4-methyl-1-oxopentan-2-yl)amino)-4-

methyl-1-oxopentan-2-yl)benzamide 7g

White crystalline solid; yield 56%; mp 187e191 ^ꢀC; R_{f (hex/EtOAc e}

¼ 0.08. IR (ATR): 3293, 3072, 2958, 2323, 1701, 1635, 1595, 1462,

3/1)

1316, 1233, 1122, 812, 705 cm^ꢁ1¹H NMR (400 MHz, DMSO‑d₆):

12.25 (1H, s, NH-Thiazole), 8.34 (2H, dd, J ¼ 8 Hz, J ¼ 9.2 Hz, NH-

J ¼ 6 Hz, CH₃-CH-CH₃). ¹³C NMR (100.61 MHz):

172.4, 172.3, 164.0,

CH-CH₂-CH-(CH₃)₂, NH-CH-CH₂-CH-(CH₃)₂), 7.83 (1H, d, J ¼ 2.8 Hz,

Ar-H), 7.62e7.51 (4H, m, Ar-H), 7.45e7.37 (4H, m, Ar-H), 7.27 (1H, d,

J ¼ 3.6 Hz, Ar-H), 5.30 (2H, s, O-CH₂-Ph), 4.65e4.54 (2H, m, NH-CH-

CH₂-CH-(CH₃)₂, NH-CH-CH₂-CH-(CH₃)₂), 1.72e1.61 (2H, m, NH-CH-

CH₂-CH-(CH₃)₂), 1.58e1.51 (1H, m, NH-CH-CH₂-CH-(CH₃)₂),

1.50e1.42 (2H, m, NH-CH-CH₂-CH-(CH₃)₂), 1.36e1.27 (1H, m, NH-

CH-CH₂-CH-(CH₃)₂), 0.96 (3H, d, J ¼ 6.4 Hz, CH₃-CH-CH₃), 0.91 (3H,

d, J ¼ 6.4 Hz, CH₃-CH-CH₃), 0.83 (3H, d, J ¼ 5.6 Hz, CH₃-CH-CH₃),

155.7, 143.1, 136.4, 132.6, 130.5, 129.2, 129.0, 128.9, 126.7, 126.7,

126.3, 125.4, 125.0, 124.1 (q, ³J (¹⁹F, ¹³C) ¼ 31.8 Hz), 119.8, 116.3, 71.5,

52.6, 52.3, 41.5, 41.0, 25.0, 24.8, 23.6, 22.1, 22.1. ¹⁹F NMR

(376.50 MHz):

(632.11): C, 62.70; H, 5.90; N, 6.65. Found: C, 62.75

e 60.3. CHN analysis: Calc. for C₃₃H₃₇ClF₃N₃O₄

0.02; H,

5.93 0.01; N, 6.79 0.02. HRMS: m/z calc. for C₃₃H₃₇ClF₃N₃O₄:

632.24975 [MþH]^þ; 654.23169 [MþNa]^þ; found: 632.25134

[MþH]^þ; 654.23308 [MþNa]^þ.

0.76 (3H, d, J ¼ 6 Hz, CH₃-CH-CH₃). ¹³C NMR (100.61 MHz):

d 172.5,

171.7, 163.9, 158.4, 155.6, 138.4, 136.4, 132.5, 130.7, 129.2, 129.0,

128.9, 125.4, 125.0, 116.2, 114.2, 71.5, 52.1, 51.7, 41.5, 40.9, 24.9, 24.8,

23.6, 22.0. CHN analysis: Calc. for C₂₉H₃₅ClN₄O₄S (571.13): C, 60.99;

H, 6.18; N, 9.81; S, 5.61. Found: C, 60.84 0.02; H, 6.31 0.02; N,

3.3.5. Characterization of 2-benzyloxy-5-chloro-N-((2S)-1-(((2S)-

1-(3,4-dichlorophenyl)amino)-4-methyl-1-oxopentan-2-yl)amino)-

4-methyl-1-oxopentan-2-yl)benzamide 7e

White crystalline solid; yield 77%; mp 197e200 ^ꢀC; R_{f (hex/EtOAc e}

9.70

0.02; S, 5.73

0.02. HRMS: m/z calc. for C₂₉H₃₅ClN₄O₄S:

¼ 0.17. IR (ATR): 3307, 3070, 2960, 1684, 1598,1586, 1498, 1301,

571.21403 [MþH]^þ; 593.19598 [MþNa]^þ; found: 571.21376

3/1)

1268, 1128, 720, 695 cm^ꢁ1¹H NMR (400 MHz, CDCl₃-d₁):

. d 8.93

[MþH]^þ; 593.19566 [MþNa]^þ.

(1H, s, NH-Ph), 8.21 (1H, d, J ¼ 4.8 Hz, NH-CH-CH₂-CH-(CH₃)₂), 8.13

(1H, d, J ¼ 2.4 Hz, NH-CH-CH₂-CH-(CH₃)₂), 7.89 (1H, d, J ¼ 2.4 Hz,

Ar-H), 7.51e7.42 (7H, m, Ar-H), 7.31 (1H, d, J ¼ 8.8 Hz, Ar-H), 7.07

(1H, d, J ¼ 8.8 Hz, Ar-H), 6.51 (1H, d, J ¼ 7.6 Hz, Ar-H), 5.18e5.12 (2H,

ABq, J ¼ 10 Hz, O-CH₂-Ph), 4.52e4.47 (1H, m, NH-CH-CH₂-CH-

(CH₃)₂), 4.38e4.33 (1H, m, NH-CH-CH₂-CH-(CH₃)₂), 1.88e1.82 (1H,

m, NH-CH-CHH-CH-(CH₃)₂), 1.61e1.44 (3H, m, NH-CH-CH₂-CH-

(CH₃)₂, NH-CH-CHH-CH-(CH₃)₂), 1.36-1-30 (1H, m, NH-CH-CH₂-

CH-(CH₃)₂), 1.19e1.11 (1H, m, NH-CH-CH₂-CH-(CH₃)₂), 0.86 (6H, dd,

J ¼ 6.4 Hz, J ¼ 26.4 Hz, CH₃-CH-CH₃), 0.73 (6H, dd, J ¼ 6.4 Hz,

3.3.8. Characterization of 2-benzyloxy-5-chloro-N-((2S)-1-(((2S)-

1-(morpholino)-4-methyl-1-oxopentan-2-yl)amino)-4-methyl-1-

oxopentan-2-yl)benzamide 7h

Light yellow solid; yield 80%; mp 55e57 ^ꢀC; R_f

(hex/EtOAc

e 1/

¼ 0.31. IR (ATR, cm^ꢁ1): 3292, 3066, 2955, 2323, 1630, 1595, 1403,

1385, 1115, 808, 695 cm^ꢁ1¹H NMR (400 MHz, DMSO‑d₆):

8.29e8.21 (2H, m, NH-CH-CH₂-CH-(CH₃)₂, NH-CH-CH₂-CH-

(CH₃)₂), 7.70 (1H, dd, J ¼ 2.8 Hz, J ¼ 4.4 Hz, Ar-H), 7.57e7.52 (3H, m,

Ar-H), 7.41e7.31 (4H, m, Ar-H), 5.28e2.22 (2H, m, O-CH₂-Ph),

4.45e4.69 (1H, m NH-CH-CH₂-CH-(CH₃)₂), 4.56e4.43 (1H, m, NH-

J ¼ 20 Hz, CH₃-CH-CH₃). ¹³C NMR (100.61 MHz, CDCl₃-d₁):

d 172.6,

R. Jorda et al. / European Journal of Medicinal Chemistry 188 (2020) 112036

CH-CH₂-CH-(CH₃)₂), 3.58e3.10 (8H, m, morpholine), 1.53e1.15 (6H,

m, NH-CH-CH₂-CH-(CH₃)_2,NH-CH-CH₂-CH-(CH₃)₂), 0.87e0.84 (6H,

m, CH₃-CH-CH₃), 0.77 (3H, t, J ¼ 5.2 Hz, CH₃-CH-CH₃), 0.71 (3H, dd,

696 cm^ꢁ1¹H NMR (400 MHz, DMSO‑d₆):

. d 10.32 (1H, s, NH-Ph),

8.63 (1H, d, J ¼ 8 Hz, NH-CH-CH₂-Ph) 8.41 (1H, d, J ¼ 7.6 Hz, NH-CH-

CH₂-Ph), 7.98 (1H, d, J ¼ 2.4 Hz, Ar-H), 7.68 (1H, d, J ¼ 3.2 Hz, Ar-H),

7.59 (1H, d, J ¼ 8.8 Hz, Ar-H), 7.52e7.49 (2H, m, Ar-H), 7.38 (2H, dd,

J ¼ 1.2 Hz, J ¼ 7.8 Hz, Ar-H), 7.35e7.23 (8H, m, Ar-H), 7.21e7.18 (1H,

m, Ar-H), 7.10 (3H, dd, J ¼ 0.8 Hz, J ¼ 4 Hz, Ar-H), 6.98 (2H, dd,

J ¼ 3.6 Hz, J ¼ 7.2 Hz, Ar-H), 5.23 (2H, t, J ¼ 12.8 Hz, O-CH₂-Ph),

4.77e4.65 (2H, m, NH-CH-CH₂-Ph, NH-CH-CH₂-Ph), 3.10 (1H, dd,

J ¼ 5.6 Hz, J ¼ 14 Hz, NH-CH-CHH-Ph), 3.03 (1H, dd, J ¼ 4.4 Hz,

J ¼ 14 Hz, NH-CH-CHH-Ph), 2.95 (1H, dd, J ¼ 9.2 Hz, J ¼ 13.6 Hz, NH-

CH-CHH-Ph), 2.66 (1H, dd, J ¼ 9.2 Hz, J ¼ 14 Hz, NH-CH-CHH-Ph).

J ¼ 6 Hz, J ¼ 11.6 Hz, CH₃-CH-CH₃). ¹³C NMR (100.62 MHz):

d 171.9,

170.6, 163.8, 155.6, 136.4, 132.5, 130.5, 129.1, 129.0, 128.9, 125.4,

125.1, 116.2, 52.2, 46.9, 46.1, 42.6, 41.8, 41.6, 41.1, 24.8, 24.7, 23.6,

22.0. CHN analysis: Calc. for C₃₀H₄₀ClN₃O₅(558.11): C, 64.56; H,

7.22; N, 7.53. Found: C, 64.31 0.03; H, 7.45 0.02; N, 6.95 0.03.

HRMS: m/z calc. for C₃₀H₄₀ClN₃O₅: 558.27293 [MþH]^þ; 580.25487

[MþNa]^þ; found: 558.27283 [MþH]^þ; 580.25467 [MþNa]^þ.

3.3.9. Characterization of 2-benzyloxy-5-chloro-N-((2S)-1-(((2S)-

1-(3,4-dichlorophenyl)amino)-4-methyl-1-oxopentan-2-yl)amino)-

1-oxo-3-phenylpropan-2-yl)benzamide 7i

¹³C NMR (100.61 MHz):

d 171.3, 171.0, 163.7, 155.5, 139.4, 137.9,

137.7,136.5,132.7,131.6,131.4,130.7,129.8,129.7,129.2,128.8,128.6,

128.4, 128.4, 127.1, 127.0, 125.6, 125.4, 124.3, 121.1, 120.0, 116.5, 71.1,

55.6, 54.9, 38.2, 38.0. CHN analysis: Calc. for C₃₈H₃₂Cl₃N₃O₄

White solid; yield 49%; mp 200e203 ^ꢀC; R_{f (hex/EtOAc e 3/1)}¼ 0.12.

IR (ATR): 3308, 3068, 2953, 1655, 1636, 1593, 1308, 1130, 1247, 715,

(701.04); C, 65.10; H, 4.60; N, 5.99. Found: C, 65.44

0.02; H,

693 cm^ꢁ1

¹H NMR (400 MHz, DMSO‑d₆):

10.18 (1H, s, NH-Ph),

4.69

0.01; N, 5.74

0.02. HRMS: m/z calc. for C₃₈H₃₂Cl₃N₃O₄:

8.38 (1H, d, J ¼ 8 Hz, NH-CH-CH₂-Ph), 8.27 (1H, d, J ¼ 7.2 Hz, NH-

CH-CH₂-CH-(CH₃)₂), 7.95 (1H, d, J ¼ 2.4 Hz, Ar-H), 7.72 (1H, d,

J ¼ 2.8, Ar-H), 7.58e7.55 (2H, m, Ar-H), 7.52e7.49 (3H, m, Ar-H),

7.37e7.32 (4H, m, Ar-H), 7.24e7.20 (4H, dd, J ¼ 2.4 Hz, J ¼ 6 Hz, Ar-

H), 7.17 (1H, d, J ¼ 6 Hz, Ar-H), 5.22 (2H, d, J ¼ 10.4 Hz, O-CH₂-Ph),

4.66e4.61 (1H, m, NH-CH-CH₂-Ph), 4.41e4.36 (1H, m, NH-CH-CH₂-

CH-(CH₃)₂), 3.08 (1H, dd, J ¼ 5.6 Hz, J ¼ 14 Hz, NH-CH-CHH-Ph),

2.91 (1H, dd, J ¼ 9.6 Hz, J ¼ 14 Hz, NH-CH-CHH-Ph), 1.30e1.23 (2H,

m, NH-CH-CHH-CH-(CH₃)₂), 1.60 (1H, dd, J ¼ 7.6 Hz, J ¼ 9.6 Hz, NH-

CH-CHH-CH-(CH₃)₂), 0.71e0.61 (6H, m, CH₃-CH-CH₃). ¹³C NMR

700.15312 [MþH]^þ; 722.13506 [MþNa]^þ; found: 700.15402

[MþH]^þ; 722.13594 [MþNa]^þ.

3.3.12. Characterization of 2-benzyloxy-5-chloro-N-((2S)-1-(((2S)-

1-(4-triﬂuoromethyl)phenyl)amino)-4-methyl-1-oxopentan-2-yl)

amino)-1-oxo-3-phenylpropan-2-yl)benzamide 7l

White solid; yield 63%; mp 212e215 ^ꢀC; R_{f (hex/EtOAc e 3/1)}¼ 0.12.

IR (ATR): 3274, 3033, 2964, 1685, 1653, 1597, 1378, 1269, 1111, 725,

695 cm^ꢁ1¹H NMR (400 MHz, DMSO‑d₆):

. d 10.30 (1H, s, NH-Ph),

8.38 (1H, d, J ¼ 7.6 Hz, NH-CH-CH₂-Ph), 8.27 (1H, d, J ¼ 7.6 Hz, NH-

CH-CH₂-CH-(CH₃)₂), 7.80 (2H, d, J ¼ 8.8 Hz, Ar-H), 7.70 (3H, dd,

J ¼ 2.8 Hz, J ¼ 16 Hz, Ar-H), 7.58e7.49 (3H, m, Ar-H), 7.39e7.32 (4H,

m, Ar-H), 7.28e7.21 (4H, m, Ar-H), 7.18e7.15 (1H, m, Ar-H), 5.04 (2H,

s, O-CH₂-Ph), 4.49 (1H, m, NH-CH-CH₂-Ph), 4.35 (1H, m, NH-CH-

CH₂-CH-(CH₃)₂), 3.09 (1H, dd, J ¼ 5.2 Hz, J ¼ 13.6 Hz, NH-CH-CHH-

Ph), 2.93 (1H, dd, J ¼ 9.6 Hz, J ¼ 14 Hz, NH-CH-CHH-Ph), 1.35e1.23

(2H, m, NH-CH-CH₂-CH-(CH₃)₂), 1.21e1.16 (1H, m, NH-CH-CH₂-CH-

(CH₃)₂), 0.69 (6H, dd, J ¼ 6 Hz, J ¼ 24 Hz, CH₃-CH-CH₃). ¹³C NMR

(100.61 MHz):

d 172.4, 171.0, 164.2, 155.7, 139.4, 138.0, 136.4, 132.7,

131.6, 131.4, 130.5, 129.8, 129.2, 129.0, 128.9, 128.7, 127.1, 125.6,

125.4, 124.8, 121.1, 119.9, 116.2, 71.5, 55.4, 52.5, 41.3, 37.8, 24.7, 23.5,

22.1. CHN analysis: Calc. for C₃₅H₃₄Cl₃N₃O₄(667.02): C, 63.02; H,

5.14; N, 6.30. Found: C, 63.03 0.02; H, 5.10 0.01; N, 6.29 0.01.

HRMS: m/z calc. for C₃₅H₃₄Cl₃N₃O₄: 666.16877 [MþH]^þ; 688.15071

[MþNa]^þ; found: 666.17021 [MþH]^þ; 688.15206 [MþNa]^þ.

3.3.10. Characterization of 2-benzyloxy-5-chloro-N-((2S)-1-(((2S)-

1-(3,4-dichlorophenyl)amino)-1-oxo-3-phenylpropan-2-yl)amino)-

4-methyl-1-oxopentan-2-yl)benzamide 7j

(100.61 MHz): d 172.2, 171.1, 164.0, 155.7, 142.9, 138.0, 136.4, 132.6,

130.6, 129.8, 129.2, 129.0, 128.9, 128.7, 127.0, 126.7, 126.3, 125.4,

124.9, 124.1 (q, J (¹⁹F, ¹³C) ¼ 32.0 Hz), 119.8, 116.2, 71.5, 55.4, 52.4,

White solid; yield 55%; mp 184e187 ^ꢀC; R_{f (hex/EtOAc e 3/1)}¼ 0.15.

IR (ATR): 3276, 3065, 2957, 1636, 1587, 1408, 1259, 1126, 714,

41.3, 37.9, 24.7, 23.5, 22.1. ¹⁹F NMR (376.50 MHz):

d e 60.3. CHN

analysis: Calc. for C₃₆H₃₅ClF₃N₃O₄(666.13): C, 64.91; H, 5.30; N,

6.31. Found: C, 65.02 0.01; H, 5.40 0.02; N, 6.36 0.02. HRMS:

m/z calc. for C₃₆H₃₅Cl F₃N₃O₄: 666.23410 [MþH]^þ; 688.21604

[MþNa]^þ; found: 666.23596 [MþH]^þ; 688.21755 [MþNa]^þ.

693 cm^ꢁ1¹H NMR (400 MHz, DMSO‑d₆):

. d 10.39 (1H, s, NH-Ph),

8.53 (2H, dd, J ¼ 8 Hz, J ¼ 13.2 Hz, NH-CH-CH₂-Ph, NH-CH-CH₂-CH-

(CH₃)₂), 8.09 (1H, d, J ¼ 1.6 Hz, Ar-H), 7.75 (1H, d, J ¼ 2.8 Hz, Ar-H),

7.71e7.60 (2H, m, Ar-H), 7.54 (1H, dd, J ¼ 2.8 Hz, J ¼ 8.8 Hz, Ar-H),

7.46 (2H, d, J ¼ 6.4 Hz, Ar-H), 7.41e7.33 (3H, m, Ar-H), 7.28 (1H, d,

J ¼ 8.8 Hz, Ar-H), 7.19e7.15 (3H, m, Ar-H), 7.08 (2H, dd, J ¼ 2.8 Hz,

J ¼ 7.6 Hz, Ar-H), 5.31 (2H, s, O-CH₂-Ph), 4.87e4.83 (1H, m, NH-CH-

CH₂-Ph), 4.56e4.50 (1H, m, NH-CH-CH₂-CH-(CH₃)₂), 3.16 (1H, dd,

J ¼ 4.4 Hz, J ¼ 14 Hz, NH-CH-CHH-Ph), 2.78 (1H, dd, J ¼ 8.8 Hz,

J ¼ 14 Hz, NH-CH-CHH-Ph), 1.71e1.60 (3H, m, NH-CH-CH₂-CH-

(CH₃)₂), 0.96 (5H, dd, J ¼ 6.4 Hz, J ¼ 17.2 Hz, CH₃-CH-CHHH), 0.85

(1H, dd, J ¼ 6.4 Hz, J ¼ 21.2 Hz, CH₃-CH-CHHH). ¹³C NMR

3.3.13. Characterization of 2-benzyloxy-5-chloro-N-((2S)-1-(((2S)-

1-(4-triﬂuoromethyl)phenyl)amino)-1-oxo-3-phenylpropan-2-yl)

amino)-4-methyl-1-oxopentan-2-yl)benzamide 7m

White solid; yield 57%; mp 201e205 ^ꢀC; R_{f (hex/EtOAc e 3/1)}¼ 0.14.

IR (ATR): 3284, 3028, 2961, 1660, 1633, 1596, 1374, 1328, 711,

693 cm^ꢁ1¹H NMR (400 MHz, DMSO‑d₆):

. d 10.43 (1H, s, NH-Ph),

8.49 (2H, dd, J ¼ 8 Hz, J ¼ 16 Hz, NH-CH-CH₂-Ph, NH-CH-CH₂-CH-

(CH₃)₂), 7.87 (2H, d, J ¼ 8.4 Hz, Ar-H), 7.70 (3H, t, J ¼ 4.4 Hz, Ar-H),

7.49 (1H, dd, J ¼ 2.8 Hz, J ¼ 8.8 Hz, Ar-H), 7.41 (2H, t, J ¼ 8.8 Hz, Ar-

H), 7.36e7.29 (3H, m, Ar-H), 7.22 (1H, d, J ¼ 9.2 Hz, Ar-H), 7.11 (3H,

dd, J ¼ 5.2 Hz, J ¼ 12 Hz, Ar-H), 7.04 (2H, dd, J ¼ 2.4 Hz, J ¼ 7.6 Hz, Ar-

H), 5.29e5.22 (2H, ABq, J ¼ 13.2 Hz, O-CH₂-Ph), 4.85e4.80 (1H, m,

NH-CH-CH₂-Ph), 4.56e4.51 (1H, m, NH-CH-CH₂-CH-(CH₃)₂), 3.12

(1H, dd, J ¼ 4 Hz, J ¼ 13.6 Hz, NH-CH-CHH-Ph), 2.73 (1H, dd,

J ¼ 9.2 Hz, J ¼ 14 Hz, NH-CH-CHH-Ph), 1.67e1.56 (3H, m, NH-CH-

CH₂-CH-(CH₃)₂), 0.91 (6H, dd, J ¼ 6.4 Hz, J ¼ 16.4 Hz, CH₃-CH-CH₃).

(100.61 MHz):

d 172.1, 171.3, 163.6, 155.5, 139.6, 137.7, 136.5, 132.7,

131.7, 131.4, 130.6, 129.7, 129.2, 128.8, 128.6, 128.4, 127.0, 125.5,

125.4, 124.5, 121.1, 120.0, 116.5, 72.1, 54.9, 52.8, 41.1, 38.2, 24.6, 23.7,

22.3. CHN analysis: Calc. for C₃₅H₃₄Cl₃N₃O₄(667.02); C, 63.02; H,

5.14; N, 6.30. Found: C, 62.99 0.02; H, 5.10 0.02; N, 6.21 0.02.

HRMS: m/z calc. for C₃₅H₃₄Cl₃N₃O₄: 666.16877 [MþH]^þ; 688.15071

[MþNa]^þ;found: 666.17036 [MþH]^þ; 688.15215 [MþNa]^þ.

3.3.11. Characterization of 2-benzyloxy-5-chloro-N-((2S)-1-(((2S)-

1-(3,4-dichlorophenyl)amino)-1-oxo-3-phenylpropan-2-yl)amino)-

1-oxo-3-phenylpropan-2-yl)benzamide 7k

¹³C NMR (100.61 MHz):

d 172.2, 171.3, 163.7, 155.5, 143.1, 137.8,

136.5, 132.6, 130.7, 129.7, 129.2, 128.8, 128.6, 128.4, 127.0, 126.7,

126.4, 125.5, 124.1 (q, ³J (¹⁹F, ¹³C) ¼ 29.5 Hz), 121.0, 119.9, 116.5, 71.1,

White solid; yield 53%; mp 224e227 ^ꢀC; R_{f (hex/EtOAc e 3/1)}¼ 0.10.

IR (ATR): 3265, 3062, 2916, 1643, 1593, 1523, 1310, 1255, 726,

54.9, 52.8, 41.2, 38.3, 25.0, 23.6, 22.3. ¹⁹F NMR (376.50 MHz):

d e

60.3. CHN analysis: Calc. for C₃₆H₃₅ClF₃N₃O₄(666.13): C, 64.91; H,

R. Jorda et al. / European Journal of Medicinal Chemistry 188 (2020) 112036

5.30; N, 6.31. Found: C, 65.19 0.02; H, 5.24 0.02; N, 6.03 0.03.

3.4.2. Characterization of 5-chloro-2-hydroxy-N-((2S)-1-(((2S)-1-

(4-methyl)phenyl)amino)-4-methyl-1-oxopentan-2-yl)amino)-4-

HRMS: m/z calc. for

₃₆H₃₅ClF₃N₃O₄: 666.23410 [MþH]^þ;

688.21604 [MþNa]^þ; found: 666.23543 [MþH]^þ; 688.21710

[MþNa]^þ.

methyl-1-oxopentan-2-yl)benzamide 8b

White solid; yield 57%; mp 215e220 ^ꢀC; R_{f (hex/EtOAc e 3/1)}¼ 0.26.

IR (ATR): 3290, 3076, 2928, 1603, 1576, 1545, 1310, 1285, 1114, 720,

690 cm^ꢁ1¹H NMR (400 MHz, DMSO‑d₆):

. d 12.26 (1H, s, OH-Ph),

3.3.14. Characterization of 2-benzyloxy-5-chloro-N-((2S)-1-(((2S)-

1-(4-triﬂuoromethyl)phenyl)amino)-1-oxo-3-phenylpropan-2-yl)

amino)-1-oxo-3-phenylpropan-2-yl)benzamide 7n

9.88 (1H, s, NH-Ph), 8.87 (1H, d, J ¼ 7.6 Hz, NH-CH-CH₂-CH-(CH₃)₂),

8.31 (1H, d, J ¼ 8 Hz, NH-CH-CH₂-CH-(CH₃)₂), 8.03 (1H, d, J ¼ 2.4 Hz,

Ar-H), 7.49e7.42 (3H, m, Ar-H), 7.10 (2H, d, J ¼ 8.4 Hz, Ar-H), 6.95

(1H, d, J ¼ 8.8 Hz, Ar-H), 4.59e4.56 (1H, m, NH-CH-CH₂-CH-(CH₃)₂),

4.49e4.43 (1H, m, NH-CH-CH₂-CH-(CH₃)₂), 2.24 (3H, s, CH₃-Ph),

1.68e1.52 (6H, m, NH-CH-CH₂-CH-(CH₃)_2,NH-CH-CH₂-CH-(CH₃)₂),

0.92e0.86 (12H, m, CH₃-CH-CH₃, CH₃-CH-CH₃). ¹³C NMR

White solid; yield 50%; mp 245e249 ^ꢀC; R_{f (hex/EtOAc e 3/1)}¼ 0.10.

IR (ATR): 3265, 3062, 2929, 1658, 1644, 1595, 1448, 1327, 1116, 719,

692 cm^ꢁ1¹H NMR (400 MHz, DMSO‑d₆):

. d 10.45 (1H, s, NH-Ph),

8.66 (1H, d, J ¼ 8 Hz, NH-CH-CH₂-Ph), 8.43 (1H, d, J ¼ 7.6 Hz, NH-

CH-CH₂-Ph), 7.84 (2H, d, J ¼ 8.4 Hz, Ar-H), 7.71 (3H, dd, J ¼ 8.8 Hz,

J ¼ 12 Hz, Ar-H), 7.31 (1H, dd, J ¼ 2.8 Hz, J ¼ 8.8 Hz, Ar-H), 7.41e7.18

(11H, m, Ar-H), 7.11 (3H, t, J ¼ 3.5 Hz, Ar-H), 7.01 (2H, t, J ¼ 4.0 Hz, Ar-

H), 5.25 (2H, s, O-CH₂-Ph), 4.81e4.73 (2H, m, NH-CH-CH₂-Ph, NH-

CH-CH₂-Ph), 3.16e2.95 (3H, m, NH-CH-CH₂-Ph, NH-CH-CHH-Ph),

2.69 (1H, dd, J ¼ 9.2 Hz, J ¼ 14 Hz, NH-CH-CHH-Ph). ¹³C NMR

(100.61 MHz):

d 172.2, 171.3, 167.2, 158.4, 137.1, 133.8, 132.9, 129.7,

128.9, 123.2, 119.9, 119.8, 118.2, 52.5, 52.3, 41.4, 41.2, 25.0, 24.9, 23.6,

22.2, 22.1. CHN analysis: Calc. for C₂₆H₃₄ClN₃O₄(488.02): C, 63.99;

H, 7.02; N, 8.61. Found: C, 64.02 0.01; H, 7.10 0.01; N, 8.52 0.02.

HRMS: m/z calc. for C₂₆H₃₄ClN₃O₄: 488.23106 [MþH]^þ; 510.21301

[MþNa]^þ; found: 488.23172 [MþH]^þ; 510.21363 [MþNa]^þ.

(100.61 MHz):

d 171.3, 171.1, 163.6, 155.5, 142.9, 137.9, 137.7, 136.5,

132.7, 130.7, 129.8, 129.7, 129.2, 128.8, 128.8, 128.6, 128.4, 127.1,

127.0, 126.8, 126.4, 125.4, 124.1 (q, ³J (¹⁹F, ¹³C) ¼ 32.0 Hz), 121.0,

3.4.3. Characterization of 5-chloro-2-hydroxy-N-((2S)-1-(((2S)-1-

(phenyl)amino)-4-methyl-1-oxopentan-2-yl)amino)-4-methyl-1-

oxopentan-2-yl)benzamide 8c

191.9, 116.5, 71.1, 55.6, 54.9, 38.2, 38.1. ¹⁹F NMR (376.50 MHz):

d e

60.3. CHN analysis: Calc. for C₃₉H₃₃ClF₃N₃O₄(700.15): C, 66.90; H,

4.75; N, 6.00. Found: C, 67.12 0.02; H, 4.72 0.01; N, 5.97 0.02.

White solid; yield 56%; mp 221e225 ^ꢀC; R_{f (hex/EtOAc e 3/1)}¼ 0.24.

IR (ATR): 3307, 3070, 2960, 1773, 1545, 1498, 1383, 1058, 820,

HRMS: m/z calc. for

₃₉H₃₃ClF₃N₃O₄: 700.21845 [MþH]^þ;

722.20039 [MþNa]^þ; found: 700.21936 [MþH]^þ; 722.20115

[MþNa]^þ.

695 cm^ꢁ1¹H NMR (400 MHz, DMSO‑d₆):

. d 12.25 (1H, s, OH-Ph),

9.96 (1H, s, NH-Ph), 8.86 (1H, d, J ¼ 8 Hz, NH-CH-CH₂-CH-(CH₃)₂),

8.32 (1H, d, J ¼ 7.6 Hz, NH-CH-CH₂-CH-(CH₃)₂), 8.03 (1H, d,

J ¼ 2.4 Hz, Ar-H), 7.60 (2H, d, J ¼ 8 Hz, Ar-H), 7.44 (1H, dd, J ¼ 2.4 Hz,

J ¼ 8.8 Hz, Ar-H), 7.30 (2H, t, J ¼ 8 Hz, Ar-H), 7.04 (1H, t, J ¼ 7.2 Hz,

Ar-H), 6.95 (1H, d, J ¼ 8.8 Hz, Ar-H), 4.60 (1H, dd, J ¼ 9.2 Hz,

J ¼ 12 Hz, NH-CH-CH₂-CH-(CH₃)₂), 4.48 (1H, dd, J ¼ 8.8 Hz,

J ¼ 14 Hz, NH-CH-CHH-CH-(CH₃)₂), 1.68e1.51 (6H, m, NH-CH-CH₂-

CH-(CH₃)₂), NH-CH-CH₂-CH-(CH₃)₂), 0.89 (12H, dd, J ¼ 6.4 Hz,

J ¼ 15.2 Hz, CH₃-CH-CH₃, CH₃-CH-CH₃). ¹³C NMR (100.61 MHz):

3.4. General procedure and characterization of debenzylated

compounds 8a-n

A solution of benzylated amide 7a-n in ethyl acetate (150 ml per

1 mmol) was hydrogenated in a three-necked round bottom ﬂask in

the presence of 10% Pd/C (0.125 mg per 1 mmol of 7) and a H₂at-

mosphere at room temperature for 16 h. The reaction was moni-

tored by TLC, and when the starting material was consumed, the

reaction was terminated. The catalyst was ﬁltered off, and the

ﬁltrate was evaporated under educed pressure. The residue was

puriﬁed using column chromatography (silica gel eluted with 20%

ethyl acetate in n-hexane), and the desired compound was ﬁnally

crystalized from an n-hexane/ethyl acetate mixture to give com-

pounds 8a-n at 35e80% of the isolated yield.

172.3, 171.6, 167.2, 158.4, 139.6, 133.8, 129.3, 128.9, 124.0, 123.2,

119.9, 119.8, 118.2, 52.6, 52.3, 41.3, 41.2, 25.0, 25.0, 23.6, 22.2. CHN

analysis: Calc. for C₂₅H₃₂ClN₃O₄(473.99): C, 63.35; H, 6.80; N, 8.87.

Found: C, 63.56 0.02; H, 6.94 0.02; N, 9.02 0.02. HRMS: m/z

calc. for C₂₅H₃₂ClN₃O₄: 474.21541 [MþH]^þ; 496.19736 [MþNa]^þ;

found: 474.21599 [MþH]^þ; 496.19789 [MþNa]^þ.

3.4.4. Characterization of 5-chloro-2-hydroxy-N-((2S)-1-(((2S)-1-

(4-triﬂuoromethyl)phenyl)amino)-4-methyl-1-oxopentan-2-yl)

amino)-4-methyl-1-oxopentan-2-yl)benzamide 8d

3.4.1. Characterization of 5-chloro-2-hyroxy-N-((2S)-1-(((2S)-1-(4-

methoxy)phenyl)amino)-4-methyl-1-oxopentan-2-yl)amino)-4-

methyl-1-oxopentan-2-yl)benzamide 8a

White crystalline solid; yield 71%; mp 207e210 ^ꢀC; R_{f (hex/EtOAc e}

White crystalline solid; yield 60%; mp 224e227 ^ꢀC; R_{f (hex/EtOAc e}

¼ 0.24. IR (ATR): 3289, 3075, 2958, 1677, 1644, 1578, 1323, 1370,

3/1)

¼ 0.17. IR (ATR): 3301, 3071, 2955, 1651, 1634, 1602, 1338, 1287,

1066, 712, 689 cm^ꢁ1. ¹H NMR (400 MHz, DMSO‑d₆):

d 12.34 (1H, s,

3/1)

1116, 735, 697 cm^ꢁ1. ¹H NMR (400 MHz, DMSO‑d₆):

12.25 (1H, s,

OH-Ph) 10.39 (1H, s, NH-Ph), 8.87 (1H, d, J ¼ 8 Hz, NH-CH-CH₂-CH-

(CH₃)₂), 8.40 (1H, d, J ¼ 7.6 Hz, NH-CH-CH₂-CH-(CH₃)₂), 8.02 (1H, d,

J ¼ 2.8 Hz, Ar-H), 7.82 (2H, d, J ¼ 8.4 Hz, Ar-H), 7.67 (2H, d, J ¼ 8.8 Hz,

Ar-H), 7.43 (1H, dd, J ¼ 2.4 Hz, J ¼ 8.8 Hz, Ar-H), 6.95 (1H, d,

J ¼ 8.8 Hz, Ar-H), 4.63e4.58 (1H, m, NH-CH-CH₂-CH-(CH₃)₂),

4.51e4.46 (1H, m, NH-CH-CH₂-CH-(CH₃)₂), 1.69e1.51 (6H, m, NH-

CH-CH₂-CH-(CH₃)₂, NH-CH-CH₂-CH-(CH₃)₂), 0.94e0.87 (12H, m,

OH-Ph), 9.83 (1H, s, NH-Ph), 8.87 (1H, d, J ¼ 7.6 Hz, NH-CH-CH₂-CH-

(CH₃)₂), 8.29 (1H, d, J ¼ 8 Hz, NH-CH-CH₂-CH-(CH₃)₂), 8.02 (1H, d,

J ¼ 2.4 Hz, Ar-H), 7.52e7.48 (2H, m, Ar-H), 7.44 (1H, dd, J ¼ 2.8 Hz,

J ¼ 8.8 Hz, Ar-H), 6.96 (1H, d, J ¼ 8.8 Hz, Ar-H), 6.89e6.86 (2H, m, Ar-

H), 4.61e4.55 (1H, m, NH-CH-CH₂-CH-(CH₃)₂), 4.47e4.41 (1H, m,

NH-CH-CH₂-CH-(CH₃)₂), 3.71 (3H, s, O-CH₃), 1.68e1.55 (6H, m, NH-

CH-CH₂-CH-(CH₃)₂, NH-CH-CH₂-CH-(CH₃)₂), 0.92e0.86 (12H, m,

CH₃-CH-CH₃, CH₃-CH-CH₃). ¹³C NMR (100.61 MHz):

d 172.4, 172.3,

CH₃-CH-CH₃, CH₃-CH-CH₃). ¹³C NMR (100.61 MHz):

172.2, 171.0,

167.2, 158.4, 143.1, 133.8, 128.9, 126.7, 126.6, 126.3, 124.1 (q, ³J (¹⁹F,

¹³C) ¼ 31.9 Hz), 123.2, 119.8, 118.2, 52.8, 52.2, 41.2, 41.1, 25.0, 25.0,

167.2,158.4,155.9,133.8,132.7,128.9,123.2,121.9,119.8,118.2,114.5,

55.8, 52.5, 52.3, 41.4, 41.2, 25.0, 24.9, 23.6, 22.1. CHN analysis: Calc.

for C₂₆H₃₄ClN₃O₅(504.02): C, 61.96; H, 6.80; N, 8.34. Found: C,

62.12 0.03; H, 6.87 0.02; N, 8.09 0.03. HRMS: m/z calc. for

23.7, 22.1. ¹⁹F NMR (376.50 MHz):

d e 60.4. CHN analysis: Calc. for

C₂₆H₃₁ClF₃N₃O₄(541.99): C, 57.62; H, 5.77; N, 7.75. Found: C,

57.63 0.02; H, 5.68 0.01; N, 7.51 0.02. HRMS: m/z calc. for

₂₆H₃₄ClN₃O₅: 504.22598 [MþH]^þ; 526.20792 [MþNa]^þ; found:

₂₆H₃₁ClF₃N₃O₄: 542.20280 [MþH]^þ; 564.18474 [MþNa]^þ; found:

504.22678 [MþH]^þ; 526.20872 [MþNa]^þ.

542.20429 [MþH]^þ; 564.18613 [MþNa]^þ.

R. Jorda et al. / European Journal of Medicinal Chemistry 188 (2020) 112036

3.4.5. Characterization of 5-chloro-2-hydroxy-N-((2S)-1-(((2S)-1-

(3,4-dichlorophenyl)amino)-4-methyl-1-oxopentan-2-yl)amino)-4-

methyl-1-oxopentan-2-yl)benzamide 8e

3.4.8. Characterization of 5-chloro-2-hydroxy-N-((2S)-1-(((2S)-1-

(morpholino)-4-methyl-1-oxopentan-2-yl)amino)-4-methyl-1-

oxopentan-2-yl)benzamide 8h

White crystalline solid; yield 85%; mp 217e220 ^ꢀC; R_{f (hex/EtOAc e}

Light yellow solid; yield 71%; mp 237e239 ^ꢀC; R_{f (hex/EtOAc e 1/}

¼ 0.22. IR (ATR): 3286, 2961, 1639, 1593, 1530, 1416, 1288, 1149,

¼ 0.26. IR (ATR): 3327, 3254, 3080, 2955, 1626, 1548, 1437, 1332,

3/1)

783, 691 cm^ꢁ1. ¹H NMR (400 MHz, DMSO‑d₆):

12.25 (1H, s, OH-

1115, 825, 697 cm^ꢁ1. ¹H NMR (400 MHz, DMSO‑d₆):

12.26 (1H, s,

Ph), 10.31 (1H, s, NH-Ph), 8.86 (1H, d, J ¼ 7.6 Hz, NH-CH-CH₂-CH-

(CH₃)₂), 8.39 (1H, d, J ¼ 7.6 Hz, NH-CH-CH₂-CH-(CH₃)₂), 8.00 (2H,

dd, J ¼ 2.4 Hz, J ¼ 14 Hz, Ar-H), 7.57e7.50 (2H, m, Ar-H), 7.43 (1H, dd,

J ¼ 2.4 Hz, J ¼ 8.8 Hz, Ar-H), 6.95 (1H, d, J ¼ 8.8 Hz, Ar-H), 4.62e4.57

(1H, m, NH-CH-CH₂-CH-(CH₃)₂), 4.42 (1H, dd, J ¼ 9.2 Hz, J ¼ 12.8 Hz,

NH-CH-CH₂-CH-(CH₃)₂), 1.68e1.48 (6H, m, NH-CH-CH₂-CH-(CH₃)₂,

NH-CH-CH₂-CH-(CH₃)₂) 0.92e0.86 (12H, m, (CH₃)-CH-(CH₃), (CH₃)-

OH-Ph), 8.84 (1H, d, J ¼ 8 Hz, NH-CH-CH₂-CH-(CH₃)₂), 8.36 (1H, d,

J ¼ 8 Hz, NH-CH-CH₂-CH-(CH₃)₂), 8.02 (1H, d, J ¼ 2.8 Hz, Ar-H), 7.45

(1H, dd, J ¼ 2.4 Hz, J ¼ 8.8 Hz, Ar-H), 6.95 (1H, d, J ¼ 8.8 Hz, Ar-H),

4.75e4.69 (1H, m, NH-CH-CH₂-CH-(CH₃)₂), 4.58e4.56 (1H, m, NH-

CH-CH₂-CH-(CH₃)₂), 3.58e3.45 (8H, m, morpholine), 1.68e1.47 (6H,

m, NH-CH-CH₂-CH-(CH₃)_2,NH-CH-CH₂-CH-(CH₃)₂), 0.91e0.73

(12H, m, CH₃-CH-CH₃, CH₃-CH-CH₃). ¹³C NMR (100.61 MHz):

d 171.8,

CH-(CH₃)). ¹³C NMR (100.61 MHz):

172.5, 172.2, 167.2, 158.4, 139.6,

170.6, 167.2, 158.5, 133.8, 128.8, 123.1, 119.8, 118.1, 66.8, 52.2, 47.2,

133.8, 131.6, 131.3, 128.9, 125.4, 123.2, 121.0, 119.9, 119.8, 118.2, 52.8,

52.2, 41.2, 41.0, 25.0, 24.9, 23.8, 23.7, 22.1 (2x C). CHN analysis: Calc.

for C₂₅H₃₀Cl₃N₃O₄(542.88): C, 55.31; H, 5.57; N, 7.74. Found: C,

55.77 0.02; H, 5.56 0.01; N, 7.72 0.01. HRMS: m/z calc. for

46.1, 42.6, 41.0, 25.0, 24.7, 23.7, 22.1. CHN analysis: Calc. for

C₂₃H₃₄ClN₃O₅(467.99): C, 59.03; H, 7.32; N, 8.98. Found: C,

60.28 0.17; H, 7.94 0.01; N, 7.97 0.02. HRMS: m/z calc. for

₂₃H₃₄ClN₃O₅: 468.22598 [MþH]^þ; 490.20792 [MþNa]^þ; found:

₂₅H₃₀Cl₃N₃O₄: 542.13747 [MþH]^þ; 564.11941 [MþNa]^þ; found:

468.22655 [MþH]^þ; 490.20850 [MþNa]^þ.

542.13882 [MþH]^þ; 564.12087 [MþNa]^þ.

3.4.9. Characterization of 5-chloro-2-hydroxy-N-((2S)-1-(((2S)-1-

(3,4-dichlorophenyl)amino)-4-methyl-1-oxopentan-2-yl)amino)-1-

oxo-3-phenylpropan-2-yl)benzamide 8i

3.4.6. Characterization of 5-chloro-2-hydroxy-N-((2S)-1-(((2S)-1-

(cyclohexyl)amino)-4-methyl-1-oxopentan-2-yl)amino)-4-methyl-

1-oxopentan-2-yl)benzamide 8f

White solid; yield 41%; mp 254e258 ^ꢀC; R_{f (hex/EtOAc e 3/1)}¼ 0.16.

IR (ATR): 3304, 2959, 1661, 1638, 1585, 1416, 1290, 1131, 733,

White solid; yield 60%; mp 195e202 ^ꢀC; R_{f (hex/EtOAc e 3/1)}¼ 0.14.

IR (ATR): 3307, 3070, 2960, 1773, 1545, 1498, 1383, 1058, 820,

697 cm^ꢁ1¹H NMR (400 MHz, DMSO‑d₆):

. d 12.29 (1H, s, OH-Ph),

10.35 (1H, s, NH-Ph), 8.88 (1H, d, J ¼ 7.6 Hz, NH-CH-CH₂-Ph), 8.56

(1H, d, J ¼ 8 Hz, NH-CH-CH₂-CH-(CH₃)₂), 8.05 (1H, d, J ¼ 2.4 Hz, Ar-

H), 7.99 (1H, d, J ¼ 2 Hz, Ar-H), 7.61 (1H, d, J ¼ 8.8 Hz, Ar-H),

7.54e7.48 (2H, m, Ar-H), 7.33e7.25 (4H, m, Ar-H), 7.20 (1H, dd,

J ¼ 4.8 Hz, J ¼ 7.2 Hz, Ar-H), 7.01 (1H, d, J ¼ 8.8 Hz, Ar-H), 4.71e4.59

(2H, m, NH-CH-CH₂-Ph, NH-CH-CH₂-CH-(CH₃)₂), 3.12 (1H, dd,

J ¼ 5.6 Hz, J ¼ 14 Hz, NH-CH-CHH-Ph), 3.00 (1H, dd, J ¼ 9.2 Hz,

J ¼ 13.6 Hz, NH-CH-CHH-Ph), 1.67e1.61 (2H, m, NH-CH-CH₂-CH-

(CH₃)₂), 1.56 (1H, dd, J ¼ 5.2 Hz, J ¼ 10.8 Hz, NH-CH-CH₂-CH-

(CH₃)₂), 0.92 (6H, dd, J ¼ 6.4 Hz, J ¼ 9.2 Hz, CH₃-CH-CH₃). ¹³C NMR

695 cm^ꢁ1¹H NMR (400 MHz, DMSO‑d₆):

. d 12.24 (1H, s, OH-Ph),

8.86 (1H, d, J ¼ 7.6 Hz, NH-cyclohexyl), 8.10 (1H, d, J ¼ 8.4 Hz, NH-

CH-CH₂-CH-(CH₃)₂), 8.02 (1H, d, J ¼ 2.4 Hz, NH-CH-CH₂-CH-

(CH₃)₂), 7.58 (1H, d, J ¼ 8 Hz, Ar-H), 7.44 (1H, dd, J ¼ 2.8 Hz,

J ¼ 8.8 Hz, Ar-H), 6.95 (1H, d, J ¼ 8.8 Hz, Ar-H), 4.50 (1H, dd, J ¼ 8 Hz,

J ¼ 12.4 Hz, NH-CH-CH₂-CH-(CH₃)₂), 4.29e4.23 (1H, m, NH-CH-

CH₂-CH-(CH₃)₂), 3.849 (1H, t, J ¼ 3.6 Hz, cyclohexyl),1.66e1.54 (10H,

m, cyclohexyl), 1.32 (6H, m, NH-CH-CH₂-CH-(CH₃)₂, NH-CH-CH₂-

CH-(CH₃)₂), 0.91e0.86 (9H, m, CH₃-CH-CH₃, CH₃-CH-CH₃), 0.81 (3H,

d, J ¼ 6.4 Hz CH₃-CH-CH₃). ¹³C NMR (100.61 MHz):

171.9, 171.3,

(100.61 MHz): d 172.3, 171.1, 167.1, 158.4, 139.4, 137.9, 133.8, 131.6,

131.4, 129.8, 128.9, 128.7, 127.1, 125.6, 123.2, 121.1, 120.0, 119.8, 118.1,

167.2, 158.4, 128.9, 125.5, 123.1, 119.8, 118.2, 52.6, 51.7, 48.0, 41.5,

41.1, 35.0, 32.9, 31.1, 25.8, 24.9, 23.7, 21.7. CHN analysis: Calc. for

55.6, 52.2, 41.0, 37.9, 25.0, 23.7, 22.2. CHN analysis: Calc. for

₂₅H₃₈ClN₃O₄(480.04): C, 62.55; H, 7.98; N, 8.75. Found: C,

C₂₈H₂₈Cl₃N₃O₄(576.9): C, 58.29; H, 4.89; N, 7.28. Found: C,

66.08 0.03; H, 8.93 0.02; N, 6.82 0.03. HRMS: m/z calc. for

58.57 0.02; H, 4.88 0.01; N, 7.02 0.02. HRMS: m/z calc. for

₂₅H₃₈ClN₃O₄: 480.26236 [MþH]^þ; 502.24431 [MþNa]^þ;found:

₂₈H₂₈Cl₃N₃O₄: 598.10376 [MþNa]^þ; found: 598.10416 [MþNa]^þ.

480.26307 [MþH]^þ; 502.24479 [MþNa]^þ.

3.4.10. Characterization of 5-chloro-2-hydroxy-N-((2S)-1-(((2S)-1-

(3,4-dichlorophenyl)amino)-1-oxo-3-phenylpropan-2-yl)amino)-4-

methyl-1-oxopentan-2-yl)benzamide 8j

3.4.7. Characterization of 5-chloro-2-hydroxy-N-((2S)-1-(((2S)-1-

(thiazol-2-yl)amino)-4-methyl-1-oxopentan-2-yl)amino)-4-

methyl-1-oxopentan-2-yl)benzamide 8g

White solid; yield 36%; mp 181e185 ^ꢀC; R_{f (hex/EtOAc e 3/1)}¼ 0.17.

IR (ATR): 3292, 3066, 2959, 1636, 1591, 1533, 1417, 1287, 1133, 744,

White crystalline solid; yield 68%; mp 206e209 ^ꢀC; R_{f (hex/EtOAc e}

698 cm^ꢁ1¹H NMR (500 MHz, DMSO‑d₆):

. d 12.03 (1H, s, OH-Ph),

¼ 0.29. IR (ATR): 3389, 3293, 2958, 2323,1545,1498, 1383,1058,

10.38 (1H, s, NH-Ph), 8.95 (1H, d, J ¼ 7.5 Hz, NH-CH-CH₂-Ph), 8.58

(1H, d, J ¼ 8 Hz, NH-CH-CH₂-CH-(CH₃)₂), 8.04 (1H, d, J ¼ 2.5 Hz, Ar-

H), 7.94 (1H, d, J ¼ 3 Hz, Ar-H), 7.60e7.55 (2H, m, Ar-H), 7.41 (1H, dd,

J ¼ 2.5 Hz, J ¼ 7.2 Hz, Ar-H), 7.28 (2H, d, J ¼ 7 Hz, Ar-H), 7.20 (2H, t,

J ¼ 7,6 Hz, Ar-H), 7.15 (1H, t, J ¼ 7.2 Hz, Ar-H), 6.93 (1H, d, J ¼ 9 Hz,

Ar-H), 4.86e4.82 (1H, m, NH-CH-CH₂-Ph), 4.47 (1H, dd, J ¼ 9.5 Hz,

J ¼ 15 Hz, NH-CH-CH₂-CH-(CH₃)₂), 3.16 (1H, dd, J ¼ 4 Hz, J ¼ 14 Hz,

NH-CH-CHH-Ph), 3.66 (1H, dd, J ¼ 9.5 Hz, J ¼ 13.5 Hz, NH-CH-CHH-

Ph), 1.69e1.60 (2H, m, NH-CH-CH₂-CH-(CH₃)₂), 1.57e1.53 (1H, m,

NH-CH-CH₂-CH-(CH₃)₂), 0.93 (3H, d, J ¼ 6.5 Hz, CH₃-CH-CH₃), 0.89

3/1)

820, 695 cm^ꢁ1¹H NMR (400 MHz, DMSO‑d₆):

. d 12.38 (2H, d,

J ¼ 20.5 Hz, OH-Ph, NH-Thiazole), 8.57 (1H, d, J ¼ 8 Hz, NH-CH-CH₂-

CH-(CH₃)₂), 8.41 (1H, d, J ¼ 7.2 Hz, NH-CH-CH₂-CH-(CH₃)₂), 8.03

(1H, dd, J ¼ 2.4 Hz, J ¼ 4.8 Hz, Ar-H), 7.47e7.42 (2H, m, Ar-H), 7.21

(1H, d, J ¼ 3.6 Hz, Ar-H), 6.95 (1H, d, J ¼ 8.8 Hz, Ar-H), 4.61e4.54 (2H,

m, NH-CH-CHH-CH-(CH₃)₂, NH-CH-CHH-CH-(CH₃)₂), 1.70e1.50

(6H, m, NH-CH-CH₂-CH-(CH₃)₂, NH-CH-CH₂-CH-(CH₃)₂), 0.90 (12H,

dd, J ¼ 1.6 Hz, J ¼ 8 Hz, CH₂-CH-CH₃, CH₂-CH-CH₃). ¹³C NMR

(100.61 MHz):

d 172.5, 171.8, 167.2, 158.4, 158.4, 138.3, 133.9, 128.8,

123.2, 119.8, 118.0, 114.2, 60.4, 52.0, 52.0, 41.1, 25.0, 25.0, 23.8, 21.9.

CHN analysis: Calc. for C₂₂H₂₉ClN₄O₄S (481.01): C, 54.93; H, 6.08; N,

11.65, S 6.67. Found: C, 55.10 0.02; H, 6.21 0.02; N, 11.49 0.02;

(3H, d, J ¼ 6.5 Hz, CH₃-CH-CH₃). ¹³C NMR (125.78 MHz):

d 172.0,

171.2, 166.4, 157.7, 139.4, 137.9, 133.6, 131.5, 131.2, 129.7, 128.8, 128.5,

126.9, 125.3, 123.0, 120.9, 119.7, 119.5, 118.2, 54.6, 52.7, 41.5, 37.7,

24.8, 23.4, 22.0. CHN analysis: Calc. for C₂₈H₂₈Cl₃N₃O₄(576.9): C,

58.29; H, 4.89; N, 7.28. Found: C, 58.57 0.02; H, 5.02 0.02; N,

7.20 0.02. HRMS: m/z calc. for C₂₈H₂₈Cl₃N₃O₄: 576.12182 [MþH]^þ;

S, 5.19

0.03. HRMS: m/z calc. for C₂₂H₂₉ClN₄O₄S: 481.16708

[MþH]^þ; 503.14902 [MþNa]^þ; found: 481.16814 [MþH]^þ;

503.14999 [MþNa]^þ.

R. Jorda et al. / European Journal of Medicinal Chemistry 188 (2020) 112036

598.10376 [MþNa]^þ; found: 576.12351 [MþH]^þ; 598.10554

[MþNa]^þ.

d 172.2, 171.2, 166.4, 157.9, 143.0, 137.9, 133.6, 129.7, 128.8, 128.6,

128.5, 126.8, 126.6, 125.9, 123.8 (q, ³J (¹⁹F, ¹³C) ¼ 25 Hz), 123.0, 119.6,

118.2, 54.6, 52.7, 41.0, 37.7, 24.8, 23.4, 22.0. ¹⁹F NMR (376.50 MHz):

3.4.11. Characterization of 5-chloro-2-hydroxy-N-((2S)-1-(((2S)-1-

(3,4-dichlorophenyl)amino)-1-oxo-3-phenylpropan-2-yl)amino)-1-

oxo-3-phenylpropan-2-yl)benzamide 8k

e 60.3. CHN analysis: Calc. for C₂₉H₂₉ClF₃N₃O₄(576.01): C, 60.47;

H, 5.07; N, 7.30. Found: C, 60.52 0.02; H, 5.25 0.01; N,

7.10

0.02. HRMS: m/z calc. for C₂₉H₂₉ClF₃N₃O₄: 576.18715

White solid; yield 47%; mp 159e162 ^ꢀC; R_{f (hex/EtOAc e 3/1)}¼ 0.12.

IR (ATR): 3263, 3085, 3025, 2946, 1647, 1595, 1545, 1339,1286, 1128,

[MþH]^þ; 598.16909 [MþNa]^þ; found: 576.18849 [MþH]^þ;

598.17021 [MþNa]^þ.

722, 698 cm^ꢁ1. ¹H NMR (400 MHz, DMSO‑d₆):

d 12.06 (1H, s, OH-

Ph), 10.41 (1H, s, NH-Ph), 8.93 (1H, dd, J ¼ 8 Hz, J ¼ 26.8 Hz, NH-CH-

CH₂-Ph), 8.71 (1H, dd, J ¼ 7.6 Hz, J ¼ 14.8 Hz, NH-CH-CH₂-Ph), 8.02

(1H, d, J ¼ 2.4 Hz, Ar-H), 7.97e7.91 (1H, m, Ar-H), 7.63 (1H, t,

J ¼ 4.8 Hz, Ar-H), 7.55 (1H, dd, J ¼ 2.4 Hz, J ¼ 8.8 Hz, Ar-H), 7.47e7.40

(1H, m, Ar-H), 7.34e7.16 (10H, m, Ar-H), 6.98e6.92 (1H, m, Ar-H),

4.89e4.84 (1H, m, NH-CH-CH₂-Ph), 4.72 (1H, dd, J ¼ 7.2 Hz,

J ¼ 13.6 Hz, NH-CH-CH₂-Ph), 3.15 (2H, dd, J ¼ 6 Hz, J ¼ 14 Hz, NH-

CH-CH₂-Ph), 3.05e3.00 (2H, m, NH-CH-CH₂-Ph). ¹³C NMR

3.4.14. Characterization of 5-chloro-2-hydroxy-N-((2S)-1-(((2S)-1-

(4-triﬂuoromethyl)phenyl)amino)-1-oxo-3-phenylpropan-2-yl)

amino)-1-oxo-3-phenylpropan-2-yl)benzamide 8n

White crystalline solid; yield 35%; mp 264e268 ^ꢀC; R_{f (hex/EtOAc e}

¼ 0.12. IR (ATR): 3315, 3267, 3085, 2923, 1650, 1628, 1596, 1371,

3/1)

1330, 1068, 724, 696 cm^ꢁ1. ¹H NMR (500 MHz, DMSO‑d₆):

d 12.03

(1H, s, OH-Ph), 10.47 (1H, s, NH-Ph), 8.92 (1H, d, J ¼ 6.5 Hz, NH-CH-

CH₂-Ph), 8.70 (1H, d, J ¼ 7.5 Hz, NH-CH-CH₂-Ph), 7.92e7.69 (5H, m,

Ar-H), 7.42e7.12 (11H, m, Ar-H), 6.91 (1H, t, J ¼ 9 Hz, Ar-H), 4.76 (2H,

m, NH-CH-CH₂-Ph, NH-CH-CH₂-Ph), 3.12e2.93 (4H, m, NH-CH-CH₂-

(100.61 MHz):

d 171.3, 171.0, 166.5, 158.0, 139.4, 137.8, 133.8, 131.6,

131.4, 129.9, 129.8, 129.0, 128.8, 128.7, 127.1, 125.6, 123.2, 121.2,

120.0, 119.7, 118.3, 117.7, 116.6, 55.7, 54.7, 40.8, 37.8. CHN analysis:

Calc. for C₃₁H₂₆Cl₃N₃O₄(610.91): C, 60.95; H, 4.29; N, 6.88. Found:

C, 63.15 0.03; H, 4.46 0.01; N, 6.76 0.02. HRMS: m/z calc. for

Ph, NH-CH-CH₂-Ph). ¹³C NMR (125.78 MHz):

d 171.1, 171.0, 166.3,

157.8, 142.7, 137.9, 137.7, 133.6, 129.7, 129.6, 128.8, 128.6, 128.5,

126.9, 126.8, 126.5, 125.9, 123.8 (q, ³J (¹⁹F, ¹³C) ¼ 25.0 Hz), 123.0,

₃₁H₂₆Cl₃N₃O₄: 632.08811 [MþNa]^þ; found: 632.08957 [MþNa]^þ.

119.7, 119.5, 118.0, 55.5, 54.5, 40.4, 37.6. ¹⁹F NMR (376.50 MHz):

d e

60.3. CHN analysis: Calc. for C₃₂H₂₇ClF₃N₃O₄(610.02): C, 63.00; H,

4.46; N, 6.89. Found: C, 63.29 0.02; H, 4.39 0.02; N, 6.68 0.02.

HRMS: m/z calc. for C₃₂H₂₇ClF₃N₃O₄: 610.17150 [MþH]^þ; 632.15344

[MþNa]^þ; found: 610.17184 [MþH]^þ; 632.15371 [MþNa]^þ.

3.4.12. Characterization of 5-chloro-2-hydroxy-N-((2S)-1-(((2S)-1-

(4-triﬂuoromethyl)phenyl)amino)-4-methyl-1-oxopentan-2-yl)

amino)-1-oxo-3-phenylpropan-2-yl)benzamide 8l

White crystalline solid; yield 52%; mp 234e238 ^ꢀC; R_{f (hex/EtOAc e}

¼ 0.14. IR (ATR): 3260, 3067, 2959, 1651, 1628, 1594, 1369, 1321,

3.5. General procedure and characterization of single amino acid-

benzyl-protected amides 9a-b

3/1)

1066, 725, 672 cm^ꢁ1. ¹H NMR (400 MHz, DMSO‑d₆):

12.28 (1H, s,

OH-Ph), 10.39 (1H, s, NH-Ph), 8.83 (1H, d, J ¼ 8 Hz, NH-CH-CH₂-Ph),

8.51 (1H, d, J ¼ 7.6 Hz, NH-CH-CH₂-CH-(CH₃)₂), 8.01 (1H, d,

J ¼ 2.4 Hz, Ar-H), 7.78 (2H, d, J ¼ 8.4 Hz, Ar-H), 7.67 (2H, d, J ¼ 8.8 Hz,

Ar-H), 7.43 (1H, dd, J ¼ 2.8 Hz, J ¼ 8.8 Hz, Ar-H), 7.28 (2H, d,

J ¼ 7.2 Hz, Ar-H), 7.22 (2H, t, J ¼ 7.6 Hz, Ar-H), 7.16 (1H, d, J ¼ 7.2 Hz,

Ar-H), 6.95 (1H d, J ¼ 8.8 Hz, Ar-H), 4.73e4.67 (1H, m, NH-CH-CH₂-

Ph), 4.61e4.56 (1H, m, NH-CH-CH₂-CH-(CH₃)₂), 3.09 (1H, dd,

J ¼ 5.2 Hz, J ¼ 14 Hz, NH-CH-CHH-Ph), 2.96 (1H, dd, J ¼ 9.2 Hz,

J ¼ 13.6 Hz, NH-CH-CHH-Ph), 1.64e1.50 (3H, m, NH-CH-CH₂-CH-

(CH₃)₂), 0.87 (6H, dd, J ¼ 6.4 Hz, J ¼ 8.4 Hz, CH₃-CH-CH₃). ¹³C NMR

EDCl∙HCl (0.97 eq) and HOBt hydrate (1.02 eq) were added to a

solution of acid 4 (1.0 eq) in dry DCM (1 mmol per 50 ml) at room

temperature. After 30 min, a solution of the 4-triﬂuromethyl aniline

(1.0 eq) in dry DCM (1 mmol per 15 ml) was added for 2 min. The

mixture was stirred at room temperature under an N₂until all

starting materials were consumed (usually 2e18 h). Afterwards, the

reaction mixture was concentrated under reduced pressure, dis-

solved in ethyl acetate (30 ml per 1 mmol) and extracted with water

2 ꢂ 10 ml per 1 mmol), sat. solution of NaHCO₃(2 ꢂ 10 ml per

1 mmol), 5% citric acid solution (2 ꢂ 10 ml per 1 mmol), brine

(1 ꢂ 10 ml per 1 mmol) and water (1 ꢂ 10 ml per 1 mmol). The

organic extract was dried with Na₂SO₄and ﬁltered and the solvent

was distilled off under vacuum. The residue was puriﬁed by column

chromatography (silica gel eluted with 25% ethyl acetate in n-

hexane) to give the benzylated product 9a-b. The obtained com-

pounds were crystallized from an n-hexane/ethyl acetate mixture

to afford the products 9a and 9b at the yields 78% and 61%,

respectively.

(100.61 MHz):

d 172.3, 171.8, 167.8, 158.4, 142.9, 137.9, 133.8, 129.8,

128.9, 128.7, 127.0, 126.7, 126.3, 124.1 (q, ³J (¹⁹F, ¹³C) ¼ 31.8 Hz),

123.2, 120.9, 119.9, 119.8, 55.5, 52.2, 41.1, 38.0, 25.0, 23.7, 22.2. ¹⁹F

NMR (376.50 MHz):

d e 60.4. CHN analysis: Calc. for

C₂₉H₂₉ClF₃N₃O₄(576.01): C, 60.47; H, 5.07; N, 7.30. Found: C,

60.59 0.01; H, 5.16 0.01; N, 7.21 0.01. HRMS: m/z calc. for

₂₉H₂₉ClF₃N₃O₄: 576.18715 [MþH]^þ; 598.16909 [MþNa]^þ; found:

576.18841 [MþH]^þ; 598.17020 [MþNa]^þ.

3.4.13. Characterization of 5-chloro-2-hydroxy-N-((2S)-1-(((2S)-1-

(4-triﬂuoromethyl)phenyl)amino)-1-oxo-3-phenylpropan-2-yl)

amino)-4-methyl-1-oxopentan-2-yl)benzamide 8m

3.5.1. Characterization of 2-benzyloxy-5-chloro-N-((2S)-1-(4-

triﬂuoromethyl)phenyl)amino)-4-methyl-1-oxopentan-2-yl)

benzamide 9a

White solid; yield 82%; mp 209e211 ^ꢀC; R_{f (hex/EtOAc e 3/1)}¼ 0.17.

IR (ATR): 3286, 3066, 2958, 1634, 1606, 1533, 1369, 1321, 1066, 727,

White solid; yield 78%; mp 113e116 ^ꢀC; R_{f (hex/EtOAc e 3/1)}¼ 0.39.

IR (ATR): 3373, 2958, 2871, 1636, 1530, 1480, 1386, 1065, 741,

697 cm^ꢁ1¹H NMR (500 MHz, DMSO‑d₆):

. d 12.10 (1H, s, OH-Ph),

10.45 (1H, s, NH-Ph), 8.96 (1H, d, J ¼ 8 Hz, NH-CH-CH₂-Ph), 8.59

(1H, d, J ¼ 8 Hz, NH-CH-CH₂-CH-(CH₃)₂), 7.94 (1H, d, J ¼ 2.5 Hz, Ar-

H), 7.87 (2H, d, J ¼ 9 Hz, Ar-H), 7.70 (2H, d, J ¼ 9 Hz, Ar-H), 7.41 (1H,

dd, J ¼ 2.5 Hz, J ¼ 8.5 Hz, Ar-H), 7.28 (2H, d, J ¼ 7 Hz, Ar-H), 7.20 (2H,

t, J ¼ 7.5 Hz, Ar-H), 7.14 (1H, t, J ¼ 7.25 Hz, Ar-H), 6.93 (1H, d, J ¼ 9 Hz,

Ar-H), 4.87e4.84 (1H, m, NH-CH-CH₂-Ph), 4.55e4.50 (1H, m, NH-

CH-CH₂-CH-(CH₃)₂), 3.18 (1H, dd, J ¼ 4 Hz, J ¼ 13.5 Hz, NH-CH-CHH-

Ph), 3.00 (1H, dd, J ¼ 9.5 Hz, J ¼ 13.5 Hz, NH-CH-CHH-Ph), 1.70e1.

(2H, m, NH-CH-CH₂-CH-(CH₃)₂), 1.59e1.56 (1H, m, NH-CH-CH₂-CH-

(CH₃)₂), 0.95e0.83 (6H, m, CH₃-CH-CH₃). ¹³C NMR (125.78 MHz):

707 cm^ꢁ1¹H NMR (400 MHz, DMSO‑d₆):

. d 10.62 (1H, s, NH-Ph),

8.47 (1H, d, J ¼ 7.6 Hz, NH-CH-CH₂-CH-(CH₃)₂), 7.87 (2H, d,

J ¼ 8.4 Hz, Ar-H), 7.78 (1H, d, J ¼ 2.8, Ar-H), 7.73 (2H, d, J ¼ 8.4 Hz, Ar-

H), 7.63e7.58 (3H, m, Ar-H), 7.46e7.39 (4H, m, Ar-H), 5.31 (2H, t,

J ¼ 12.4 Hz, O-CH₂-Ph), 4.70e4.64 (1H, m, NH-CH-CH₂-CH-(CH₃)₂),

1.52e1.42 (3H, m, NH-CH-CH₂-CH-(CH₃)₂), 0.89e0.84 (3H, m, CH₃-

CH-CH₃), 0.80 (3H, d,

5.6 Hz, CH₃-CH-CH₃). ¹³C NMR

(100.61 MHz): 172.1, 164.2, 155.7, 143.1, 136.4, 132.6, 130.5, 129.2,

129.0, 128.9, 126.7, 126.3, 125.4, 124.8, 124.4 (q, ³J

(

¹⁹F,

¹³C) ¼ 31.7 Hz), 119.9, 116.2, 71.6, 53.2, 41.3, 25.0, 23.5. ¹⁹F NMR

R. Jorda et al. / European Journal of Medicinal Chemistry 188 (2020) 112036

(376.50 MHz):

(518.96): C, 62.49; H, 5.05; N, 5.40. Found: C, 62.71

5.31 0.01; N, 5.22 0.02. HRMS: m/z calc. for C₂₇H₂₆ClF₃N₂O₃:

519.16568 [MþH]^þ; 541.14763 [MþNa]^þ; found: 519.16689

[MþH]^þ; 541.14878 [MþNa]^þ.

e 60.4. CHN analysis: Calc. for C₂₇H₂₆ClF₃N₂O₃

0.03; H,

3.6.2. Characterization of 5-chloro-2-hydroxy-N-((2S)-1-(4-

triﬂuoromethyl)phenyl)amino)-1-oxo-3-phenylpropan-2-yl)

benzamide 10b

White crystalline solid; yield 77%; mp 218e222 ^ꢀC; R_{f (hex/EtOAc e}

¼ 0.33. IR (ATR): 3371, 3270, 3091, 2956, 1686, 1532, 1546, 1372,

3/1)

1068, 826, 696 cm^ꢁ1. ¹H NMR (400 MHz, DMSO‑d₆):

d 12.05 (1H, s,

OH-Ph), 10.65 (1H, s, NH-Ph), 9.12 (1H, d, J ¼ 7.6 Hz, NH-CH-CH₂-

Ph), 7.98 (1H, d, J ¼ 2.4 Hz, Ar-H), 7.81 (2H, d, J ¼ 8.8 Hz, Ar-H), 7.70

(2H, d, J ¼ 8.4 Hz, Ar-H), 7.43 (1H, dd, J ¼ 2.4 Hz, J ¼ 8.8 Hz, Ar-H),

733-7.18.50 (5H, m, Ar-H), 6.95 (1H, d, J ¼ 8.8 Hz, Ar-H), 4.94 (1H, dd,

J ¼ 8.4 Hz, J ¼ 12.8 Hz, NH-CH-CH₂-Ph), 3.22 (1H, dd, J ¼ 5.2 Hz,

J ¼ 14 Hz, NH-CH-CHH-Ph), 3.12 (1H, dd, J ¼ 9.2 Hz, J ¼ 13.6 Hz, NH-

3.5.2. Characterization of 2-benzyloxy-5-chloro-N-((2S)-1-(4-

triﬂuoromethyl)phenyl)amino)-1-oxo-3-phenylpropan-2-yl)

benzamide 9b

White crystalline solid; yield 61%; mp 182e185 ^ꢀC; R_{f (hex/EtOAc e}

¼ 0.29. IR (ATR): 3269, 3084, 3022, 1631, 1609, 1497, 1375, 1066,

3/1)

750, 692 cm^ꢁ1. ¹H NMR (400 MHz, DMSO‑d₆):

10.60 (1H, s, NH-

CH-CHH-Ph). ¹³C NMR (100.62 MHz):

d 170.9, 166.8, 158.0, 142.9,

Ph), 8.58 (1H, d, J ¼ 7.6 Hz, NH-CH-CH₂-Ph), 7.80 (2H, d, J ¼ 8.8 Hz,

Ar-H), 7.68 (1H, s, Ar-H), 7.67 (2H, d, J ¼ 2.8 Hz, Ar-H) 7.51 (1H, dd,

J ¼ 2.8 Hz, J ¼ 9.2 Hz, Ar-H), 7.47 (2H, t, J ¼ 7.2 Hz, Ar-H), 7.39e7.31

(3H, m, Ar-H), 7.26 (1H, d, J ¼ 9.2 Hz, Ar-H), 7.21e7.14 (3H, m, Ar-H),

7.07 (2H, t, J ¼ 1.2, 6.8 Hz, Ar-H), 5.27 (2H, s, O-CH₂-Ph), 4.93e4.87

(1H, m, NH-CH-CH₂-Ph), 3.11 (1H, dd, J ¼ 5.2 Hz, J ¼ 14 Hz, NH-CH-

CHH-Ph), 2.82 (1H, dd, J ¼ 9.2 Hz, J ¼ 14 Hz, NH-CH-CHH-Ph). ¹³C

137.8, 133.9, 129.8, 129.0, 128.9, 127.2, 126.8, 124.2 (q, ³J (¹⁹F,

¹³C) ¼ 31.7 Hz), 123.6, 123.3, 120.0, 119.8, 118.1, 56.0, 37.9. ¹⁹F NMR

(376.50 MHz):

d e 60.4. CHN analysis: Calc. for C₂₃H₁₈ClF₃N₂O₃

(462.85): C, 59.68; H, 3.92; N, 6.05. Found: C, 60.06

0.03; H,

4.02 0.01; N, 6.01 0.02. HRMS: m/z calc. for C₂₃H₁₈ClF₃N₂O₃:

463.10308 [MþH]^þ; 485.08503 [MþNa]^þ; found: 463.10398

[MþH]^þ; 485.08595 [MþNa]^þ.

NMR (100.61 MHz):

d 170.9, 164.0, 155.6, 142.9, 137.5, 136.5, 132.7,

130.5, 129.7, 129.2, 129.0, 128.6, 127.2, 126.7, 126.7, 126.3, 125.5,

3.7. Biological evaluation

124.5, 123.9 (q, ³J (¹⁹F, ¹³C) ¼ 31.8 Hz), 120.0, 116.5, 71.3, 56.0, 38.2.

¹⁹F NMR (376.50 MHz):

60.3. CHN analysis: Calc. for

3.7.1. Cancer cell lines and cytotoxicity assay

₃₀H₂₄ClF₃N₂O₃(552.97): C, 65.16; H, 4.37; N, 5.07. Found: C,

Human cancer cell lines were obtained from ECACC and culti-

vated according to the provider’s instructions. In brief, cell lines

were maintained in DMEM or RPMI8226 medium supplemented

with foetal bovine serum, penicillin (100 U/mL), and streptomycin

(100 mg/mL) at 37 ^ꢀC in 5% CO₂. The cells were assayed with

compounds using three-fold dilutions in triplicate. Treatment las-

ted for 72 h, after which Calcein AM solution was added and the

ﬂuorescence of live cells was measured at 485 nm/538 nm (ex/em)

with a Fluoroskan Ascent microplate reader (Labsystems). The GI₅₀

value, the drug concentration lethal to 50% of the tumour cells, was

calculated from the obtained dose response curves.

65.40 0.02; H, 4.32 0.01; N, 4.99 0.02. HRMS: m/z calc. for

₃₀H₂₄ClF₃N₂O₃: 553.15003 [MþH]^þ; 575.13198 [MþNa]^þ; found:

553.15131 [MþH]^þ; 575.13314 [MþNa]^þ.

3.6. General procedure and characterization of debenzylated

compounds 10a-b

A solution of benzylated amide 9a-b in ethyl acetate (150 ml per

1 mmol) was hydrogenated in three necked round bottom ﬂask in

the presence of 10% Pd/C (0,125 mg per 1 mmol of 9) and H₂at-

mosphere at room temperature for 24 h. The reaction was moni-

tored by TLC, and when the starting material was consumed, the

reaction was terminated. Then, the catalyst was ﬁltered off, and the

ﬁltrate was evaporated under reduced pressure. The residue was

puriﬁed using column chromatography (silica gel eluted with 20%

ethyl acetate in n-hexane). Finally, the desired compound was

crystallized from an n-hexane/ethyl acetate mixture to give com-

pounds 10a and 10 b at the yields of 78% and 77%, respectively.

3.7.2. Time lapse microscopy

For time lapse microscopy, HCT-116 cells were plated at the

appropriate density of 10 000 cells/well in 96-plates and treated

with different compounds or different concentrations of tested

compounds for the indicated times. IncuCyte® Cytotox Red Reagent

(#4632) was added for the real-time evaluation of cell membrane

integrity and cell death according to the manufacturer’s in-

structions. Time lapse images were captured by IncuCyte ZOOM®

(Essen BioScience). Images were acquired (1 image per hour) in a

red channel and subsequent integrated software analysis was used

for cell quantiﬁcation. Cell proliferation was monitored by ana-

lysing the occupied area (% conﬂuence) of cell images over time

with conﬂuence image mask (Gold) using the IncuCyte™ cell pro-

liferation assay.

3.6.1. Characterization of 5-chloro-2-hydroxy-N-((2S)-1-(4-

triﬂuoromethyl)phenyl)amino)-4-methyl-1-oxopentan-2-yl)

benzamide 10a

White crystalline solid; yield 78%; mp 161e164 ^ꢀC; R_{f (hex/EtOAc e}

¼ 0.44. IR (ATR): 3362, 3274, 3138, 2962, 1671, 1637, 1435, 1323,

3/1)

1066, 842, 650 cm^ꢁ1. ¹H NMR (500 MHz, DMSO‑d₆):

12.33 (1H, s,

3.7.3. Caspase-3/7 assay

OH-Ph), 10.67 (1H, s, NH-Ph), 9.10 (1H, d, J ¼ 7.5 Hz, NH-CH-CH₂-

CH-(CH₃)₂), 8.13 (1H, d, J ¼ 3 Hz, Ar-H), 7.90 (2H, d, J ¼ 8.5 Hz, Ar-H),

7.74 (2H, d, J ¼ 8.5 Hz, Ar-H), 7.51 (1H, dd, J ¼ 2.5 Hz, J ¼ 8.5 Hz, Ar-

H), 7.02 (1H, d, J ¼ 9 Hz, Ar-H), 4.80e4.75 (1H, m, NH-CH-CH2-CH-

(CH₃)₂), 1.88e1.65 (3H, m, NH-CH-CH₂-CH-(CH₃)₂), 0.99 (6H, dd,

The cells were homogenized in an extraction buffer (10 mM KCl,

5 mM HEPES, 1 mM EDTA, 1 mM EGTA, 0.2% CHAPS, protease in-

hibitors, pH 7.4) on ice for 20 min. The homogenates were sepa-

rated by centrifugation at 10 000ꢂg for 30 min at 4 ^ꢀC, and then the

proteins were quantiﬁed and diluted to equal concentrations. Next,

the lysates were incubated for 4 h with 100 mM Ac-DEVD-AMC, as

the substrate of caspases 3 and 7, in the assay buffer (25 mM PIPES,

2 mM EGTA, 2 mM MgCl₂, 5 mM DTT, pH 7.3). The ﬂuorescence of

the product was measured using a Fluoroskan Ascent microplate

reader (Labsystems) at 355/460 nm (excitation/emission).

J ¼ 7 Hz, J ¼ 14.5 Hz, CH₃-CH-CH₃). ¹³C NMR (125.76 MHz):

d 171.8,

167.2, 158.2, 142.8, 133.8, 128.6, 126.5, 126.5, 125.8, 124.0 (q, ³J (¹⁹F,

¹³C) ¼ 26 Hz), 123.0, 119.6, 117.5, 53.0, 40.7, 24.9, 21.8. ¹⁹F NMR

(376.50 MHz):

d e 60.4. CHN analysis: Calc. for C₂₀H₂₀ClF₃N₂O₃

(428.83): C, 56.02; H, 4.70; N, 6.53. Found: C, 56.20

0.01; H,

4.67 0.01; N, 6.50 0.02. HRMS: m/z calc. for C₂₀H₂₀ClF₃N₂O₃:

429.11873 [MþH]^þ; 451.10068 [MþNa]^þ; found: 429.11958

[MþH]^þ; 451.10154 [MþNa]^þ.

3.7.4. Cell cycle analysis

Asynchronous cells were treated with different concentrations of

R. Jorda et al. / European Journal of Medicinal Chemistry 188 (2020) 112036

compound for the indicated time. The cultures were fed and pulse-

labelled with 10

M 5-bromo-2⁰-deoxyuridine (BrdU) for 30 min at

doi.org/10.1016/j.ejmech.2020.1 12036.

37 ^ꢀC before harvesting. The cells were trypsinized, washed with

phosphate-bufferedsalinecontaining1%bovineserumalbumin(PBS/

BSA), ﬁxed with ice-cold 70% ethanol, incubated on ice for 30 min,

washed with PBS/BSA again and resuspended in 2N HCl for 30 min at

room temperature to denature DNA. After neutralization with 0.1 M

Na₂B₄O₇, the cells were harvested by centrifugation and washed with

PBS/BSA containing 0.5% Tween-20. The cells were then stained with

an anti-BrdU ﬂuorescein-labelled antibody (1:50, eBioscience) for

1hatroomtemperatureinthedark, washedwithPBS, incubatedwith

propidium iodide for 1 h at room temperature in the dark, and ﬁnally

analysed by ﬂowcytometry using a 488 nm laser (BD FACS Verse with

software BD FACSuite, version 1.0.6.). At least 10 000 events were

acquired per each sample.

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Appendix A. Supplementary data

Supplementary data to this article can be found online at https://

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